FI103984B - A method for improving and regulating enzyme activity using noble gases - Google Patents

Description

103984

Method for Enhancing and Adjusting Enzymatic Activity Using Inert Gases Förfarande för att befrämja och reglera enzymaktivitet 5 med ädelgaser

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The present invention relates to a method for improving and controlling enzyme activity by means of noble gases.

The ability of helium (He), neon (Ne), argon (Ar), krypton (Kr), xenon (Xe) and radon (Ra) to combine in chemical combinations with other atoms is very limited. In general, only krypton, xenon and radon have been reacted with other atoms such as fluorine and oxygen, and the compounds thus formed are explosively unstable. See Advanced Inorganic Chemistry, F.A. Cotton and G. Wilkinson (Wiley, Third Edition). Although noble gases are generally chemically inert, xenon is known to have certain physiological effects, such as anesthetics. Other physiological effects have also been observed with other inert gases, such as nitrogen, which, for example, are known to cause anesthesia when used under high pressure in deep sea diving.

U.S. Patent 3,183,171 (Schreiner) discloses that argon and other inert gases can affect the rate of growth of fungi and argon is known to improve the preservation of fish or seafood. US 4,946,326 (Schvester), JP 52105232, JP 80002271 and JP 77027699. However, the lack of basic understanding of these findings renders the results difficult, if not impossible, to interpret. In addition, the purpose of these observations is further obscured by the knowledge that mixtures of many gases, including oxygen, were used in these studies. In addition, some of these studies were conducted under hyperbaric (above atmospheric pressure) pressures and freezing temperatures. At such high pressures, it is likely that the results observed were based on pressure damage caused by cellular components and the enzymes themselves.

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For example, between 1964 and 1966, Schreiner presented the physiological effects of inert gases, in particular related to anesthetic effects and studies related to the development of suitable embedded atmospheres for deep sea diving, submarines and spacecraft. The results of this study have been summarized in 9 to 3 reports, each entitled "Technical Report. The Physiological Effects of the Argon, Helium and the Rare Gases," prepared by the Office of Naval Research, Department of the Navy. Contract Nonr 4115 (00), NR: 102-597.

Three subsequent summaries and abstracts of this study were published.

10 One Summary, "Inert Gas Interactions and Effects on Enzymatically Active Proteins", Fed. Proc. 26: 650 (1967), reiterates the finding that noble and other inert gases produce physiological effects at elevated partial pressures in intact animals (anesthesia) and in microbiological and mammalian cell systems (growth inhibition).

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Another summary, A Possible Molecular Mechanism for the Biological Activity of Chemically Inert Gases, "In: Intern. Congr. Physiol. Sci., 23rd, Tokyo, again reveals that inert gases have different levels of biological activity at high pressures in the cellular system. .

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Schreiner also published a summary of the general biological effects of noble gases, in which the principal results of his earlier research are presented. General Biological Effects of the Helium-Xenon Series of Elements, Fed. Proc., 27: 872-878 (1968).

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However, in 1969, Behnke et al., Reversed most of Schreiner's conclusions.

Behnke et al came to the conclusion that the effects previously presented by Schreiner are not reproducible and are merely the result of hydrostatic pressure, i. that no effect of noble gases on enzymes can be demonstrated.

30 Enzyme-Catalyzed Reactions as Influenced by Inert Gases at High Pressures. J.

Food Sci. 34: 370-375.

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Essentially, Schreiner's studies were based on the assumption that chemically inert gases compete with oxygen molecules at cell sites and that oxygen substitution depends on the ratio of oxygen to inert gas concentrations. This assumption was never made, since the highest observed efficiencies (only inhibitory effects were observed) were observed with nitric oxide and were found to be independent of the partial oxygen pressure. In addition, the inhibition observed was only 1.9% inhibition per atmosphere of added nitric oxide.

To override Schreiner's previous work, Behnke et al independently tested the effect of high hydrostatic pressures on enzymes and sought to replicate Schreiner's results. Behnke et al found that when the pressure of nitrogen or argon gas was raised above that necessary to detect weak inhibition of chymotrypsin, invertase and tyrosinase, the inhibition did not increase, contrary to what Schreiner had observed.

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The observations of Behnke et al can be explained by a simple initial hydrostatic inhibition which is released when the pressure stabilizes. Clearly, the observations cannot be explained by the dependence of chemically inert C> 2 gas, as proposed by Schreiner. Behnke et al concluded that high pressure inert gases inhibit tyrosinase in non-fluid (i.e., gelatin) systems by reducing: access to oxygen rather than physically altering the enzyme. This conclusion is the opposite of Schreiner's observations.

In addition to the annulment by Behnke et al., The results presented by Schreiner are difficult to interpret, if not impossible, for other reasons.

First, all analyzes were performed at very high pressure and the effects of hydrostatic pressure were not controlled.

30 Secondly, for many purposes, no significant differences were found between the different noble gases and between the noble gases and nitrogen.

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Third, knowledge of the enzyme activity and inhibition model during these studies was very limited, as were the purities of the enzymes used. It is impossible to be sure that there were no interfering enzyme effects or that the measurements were made at a level of resolution sufficient to assess the effects of the various gases. In addition, all specific influence models could only be presented as an untested assumption.

Fourth, the differences in solubility of the various gases were not controlled or even considered in the result.

Fifth, all tests were performed using high pressures of inert gases, superimposed at atmospheric pressure of one atmosphere, and thus insufficient control of the oxygen pressure.

Sixth, only the inhibitory effects of gases were shown.

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Seventh, not all work procedures were fully described and may not have been experimentally controlled. In addition, long delays in the initiation of the enzyme reaction were ruled out by monitoring the overall event of the reaction, resulting in the most easily detectable rate of change.

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Eighth, the data presented varied greatly based on the small number of observations m, and thus the results are not very significant.

In summary, the inhibition values observed are very low even at high pressures.

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Tenth, studies showing a dependence on enzyme concentration * do not provide important useful patterns.

Nineteen, all reports of the inhibitory property of inert gases at low pressures, m.p. <2 atm, are assumed by extrapolation of lines obtained from measurements taken at high pressures and are not actual data.

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Finally, it is worth reiterating the fact that Bahnke et al.'s results are clearly the opposite of those presented by Schreiner in several crucial respects, mainly that the effects of high pressure are small and that the hydrostatic effects not controlled by Schreiner are primarily erroneous conclusions w 5 caused by these studies.

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In addition, while Sandhoff et al., FEBS Letters, vol. 62, No. 3 (March, 1976) suggested that xenon, nitric oxide, and halothane contribute to the action of particulate sialidase, these results are questionable depending on the use of highly impure enzymes in this study and are likely due to the inhibitory oxidases present in the particles.

The following is a summary of the foregoing patents and publications, and other related references.

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Behnke et al (1969) disclose that inert gases under high pressure affect enzyme-catalyzed reactions. J.Food Sci. 34: 370-375.

Schreiner et al (1967) describe the interactions and effects of sea gas on enzymatically active proteins. Abstract No. 2209. Fed. Proc., 26: 650.

Schreiner, H.R. 1964, Technical Report, describes the physiological effects of argon, helium and rare gases. Contract Nonr 4115 (00), NR: 102-597. Office of Naval Research, Washington, D.C.

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Schreiner, H.R. 1965, Technical Report, discloses the physiological effects of argon, helium and rare * gases. Contract Nonr 4115 (00), NR: 102-597. Office of

Naval Research, Washington, D.C.

30 Schreiner, H.R. 1966, Technical Report, discloses the physiological effects of argon, helium and rare gases. Contract Nonr 4115 (00), NR: 102-597. Office of Naval Research, Washington, D.C.

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It is important to know that none of the above studies have reached the empirical (= experimental) conclusion that the treatment gases interact with the active sites of the enzymes.

Nevertheless, it is currently known that there are many ways to inhibit the effects of an enzyme. For example, many enzymes can be inhibited by specific toxins; which may be structurally related to their normal substrates. Alternatively, many different reagents are known to be specific inactivators of target enzymes. These reagents generally cause chemical changes in the enzyme-10 min active site, resulting in loss of catalytic activity, unaltered inactivation of the active sites, or change in affinity value. See Enzymatic Reaction Mechanisms C. Walsh (W.H. Freeman & Co., 1979). Alternatively, several multi-enzyme sequences are known to be regulated by specific enzymes known as regulatory or allosteric enzymes. See 15 Bioenergetics A.L. Leninger (Benjamin / Cummings Publishing Co., 1973).

However, it would be highly advantageous if a much simpler approach could be obtained to regulate enzyme effects in a predictable and controlled manner. Furthermore, it would be highly advantageous to find a way to selectively inhibit or ameliorate enzyme effects in a predictable or controllable manner.

Thus, it is an object of the present invention to provide a method for controlling enzyme effects in a simple and direct manner.

It is also an object of the present invention to provide a method for controlling enzyme effects by using reagents structurally engineered to inactivate target enzymes.

It is also an object of the present invention to provide a method for regulating enzyme effects using enzyme toxins having a structure associated with normal enzyme substrates.

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It is a further object of the present invention to provide a method for altering optimal relative enzyme substrate concentrations and modifying optimal reaction conditions with respect to physical parameters.

The foregoing objects and other objects will become more apparent from the following description, a method for controlling enzyme effects, wherein one or more enzymes are contacted with a gas comprising one or more noble gases.

Figure 1 depicts the absorption spectrum of tyrosinase.

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Figure 2 illustrates the absorption spectrum of L-tyrosine.

Figure 3 depicts the absorption spectrum of L-tyrosine-modified tyrosinase and L-tyrosine.

Figure 4 illustrates an overview of the results of experimental runs obtained at various concentrations of tyrosinase showing a direct linear dependence of first order tyrosinase concentration.

Figure 5 shows inhibition of tyrosinase with xenon at the same weight ratio p / p.

20th Figure 6 shows very high inhibition of tyrosinase by xenon at 26 ° C.

Figure 7 illustrates that at 15 ° C, argon weakly inhibits the balance of the tyrosinase L-tyrosine reaction * 25; xenon has a small but significant inhibitory effect and neon and krypton have a greater inhibitory effect.

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Figure 8 illustrates the inhibitory effects of xenon, krypton, neon and argon on the tyrosinase L-tyrosine reaction at 20 ° C.

Figure 9 illustrates that oxygen has no cure effect on the tyrosinase L-tyrosine reaction at 20 ° C.

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Figure 10 illustrates the inhibitory effect of neon, argon, krypton and xenon on the tyrosinase L-tyrosine reaction at 25 ° C.

Figure 11 illustrates that oxygen has no healing effect on the tyrosinase-L-ty-5 rosin reaction at 25 ° C.

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Figure 12 illustrates the inhibitory effect of neon, argon, krypton and xenon on the tyrosinase L-tyrosine reaction at 30 ° C.

Figure 13 illustrates that oxygen has no healing effect on the tyrosinase L-tyrosine reaction at 30 ° C.

Figure 14 depicts standard air travel at various temperatures and shows that rate changes are directly related to differences in oxygen solubility.

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Figure 15 illustrates the inhibitory effect of neon on the tyrosinase L-tyrosine reaction, which is positively temperature dependent.

Figure 16 depicts a direct inverse (negative) relationship between krypton tyrosinase inhibitory ability and temperature.

Figure 17 illustrates the inhibition of tyrosinase L-tyrosine balance by xenon inversely temperature dependent.

Figure 18 illustrates that at 20 ° C, oxygen does not improve the tyrosinase-L-ty-rosin reaction, whereas argon, xenon and neon each dramatically inhibit the reaction. Krypton has a lower inhibitory effect than nitrogen

Figure 19 illustrates that at 25 ° C, argon and nitrogen inhibit tyrosinase L-tyrosine less than noble gases and oxygen promotes the reaction.

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Figure 20 illustrates that at 30 ° C, oxygen no longer promotes the tyrosinase L-tyrosine reaction due to lower solubility.

Figure 21 illustrates inhibition of the tyrosinase L-tyrosine reaction by neon at various temperatures 5 and shows a transition temperature in the range of 20 ° C and 25 ° C.

Figure 22 illustrates the inhibition of the tyrosinase L-tyrosine reaction with argon at various temperatures.

Figure 23 illustrates the inhibition of the tyrosinase L-tyrosine reaction by krypton at various temperatures.

Figure 24 illustrates the inhibition of the tyrosinase L-tyrosine reaction by xenon at various temperatures.

Figure 25 illustrates the inhibition of the tyrosinase L-tyrosine reaction only by a solubility mechanism involving the removal of oxygen from the solution.

Figure 26 illustrates the inhibition of tyrosinase activity by nitrogen.

Figure 27 illustrates the large difference in xenon effect between 20 ° and 25 ° C.

Figure 28 depicts glucose oxidase inhibition by krypton, xenon, argon, nitrogen and neon.

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Figure 29 illustrates the inhibition of α-glutamyltranspeptidase by krypton, xenon, argon and a mixture of krypton and xenon.

Figure 30 illustrates the inhibition of aspartate aminotransferase by krypton, xenon, argon and a mixture of krypton and xenon.

Figure 31 illustrates the promotion of α-D-glucosidase with nitrogen, neon and oxygen.

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Figure 32 illustrates the enhancement of phenylalanine ammonia lyase by neon, oxygen and nitrogen.

Figure 33 illustrates the promotion of citrate synthase with xenon and krypton and its inhibition with argon.

Figure 34 illustrates the improvement of phosphoglucose isomerase with xenon, krypton, argon and a mixture of krypton and xenon at 10 ° C.

Figure 35 illustrates the improvement of phosphoglucose isomerase with neon and nitrogen and its inhibition by oxygen at 25 ° C.

Figure 36 illustrates the promotion of S-acetyl CoA synthetase with krypton and a mixture of krypton and xenon at 25 ° C.

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Figure 37 depicts the inhibitory effect of air and nitrogen on D-glucosidase at a concentration of 100 atm. at 25 ° C.

Figure 38 illustrates the inhibitory effect of air and nitrogen at 30 atmospheric pressure of B-glucosidase.

Figure 39 depicts the inhibitory effect of air, xenon and nitrogen on tyrosinase at 30 atmospheric pressure.

Figure 40 depicts the inhibitory effect of air, xenon and nitrogen on tyrosinase at 100 atmospheric pressure.

Figure 41 illustrates the effect of enzyme substrate concentrations on noble gas enhancement / inhibition results.

Figure 42 illustrates the differential enhancement effects of xenon and neon, then by inhibiting lactate dehydrogenase at 10 ° C, for example.

30 11 103984

Figure 43 illustrates that even at 60 ° C, noble gases have a promotional or inhibitory effect on enzymes.

Figure 44 illustrates the healing effect of xenon β-glucosidase in immobilized form.

Figure 45 depicts uv / vis (ultraviolet light / visible light) absorption curves for β-glucosidase in air at five different substrate concentrations.

Figure 46 depicts uv / vis absorbance curves for β-glucosidase in xenon at five different substrate concentrations.

Fig. 47 depicts linear changes in the first order power curve regression rate of β-glucosidase air gasified reactions.

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Figure 48 depicts linear changes in the first-order power curve regression rate of β-glucosidase xenon reactions.

Figures 49 and 50 depict the same first-order velocity approximation-20 gressiolinearisations for all gases studied at this time.

Figures 51 and 52 depict data for the first 160 seconds of individual tyrosinase assays, expressed as power curves for air, then xenon contamination.

In accordance with the present invention, it has now surprisingly been found that enzyme effects can be controlled in a controlled and predictable manner by contacting one or more enzymes with a gas containing one or more noble gases. Quite surprisingly, noble gases have been found to have significant effects on enzymes even at low pressure and over a wide temperature range.

In fact, the present invention is very surprising for a number of reasons. First, according to the present invention, the regulation of enzyme effects can be accomplished at low pressures. However, even higher pressures may be used. Secondly, mixtures of noble gases, which may contain other gases such as nitrogen, oxygen, carbon dioxide, carbon monoxide and nitric oxide, for example, also give excellent results. In general, any gases can be used in addition to the noble gases 5 present in the gas mixture. However, the most typical of such other gases are oxygen, nitrogen and carbon dioxide.

The term "noble gas" as used herein refers to any gas of the group helium, neon, argon, krypton, xenon or radon. However, it is generally preferred to use neon, argon, krypton, or xenon simply because helium has low solubility and is rapidly depleted of application systems and radon is dangerously radioactive.

According to the present invention, a single pure noble gas or mixtures of noble gases may be used. For example, it is preferable to use inexpensive off-gas gases having a composition of about 90% Kr and 10% by volume Xe based on the total gas volume. However, for example, a mixture of one or more noble gases with other gases such as nitrogen, sulfur hexafluoride, carbon dioxide or oxygen may be used.

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According to the present invention, it has been found that the effects of one or more noble gases on the action of the enzyme are observed in aqueous or organic solutions, dispersions, suspensions, or other types of matrices such as gels. In addition, the regulated enzymes may or may not be bound to carriers. The noble gases themselves can be in solution, atmosphere or in bound form.

The present invention proceeds from the basic observation that the effect of enzymes representing all six classes of enzymes, classified by the Joint Commission on Biochemical Nomenclature and International Union of Pure and Applied Chemistry, is reproducibly enhanced or inhibited even upon contact. with pure noble gases or mixtures thereof, or even mixtures thereof with other gases, even at low pressures.

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According to the present invention, it has been found that the gases and gas mixtures of the present invention can regulate the action of the enzyme both kinetically and thermodynamically. Thus, it is now possible to control both the rate and yield of the enzyme reaction.

5

Although the solubility of the present gases and gas mixtures in the reaction mixtures affects the measured effect of the gases and gas mixtures, the effect is not completely determined by the solubility of the gases. Most of the effect is due to molecular effects such as polarity, ionization, Van der Waals forces and atomic rays. In general, according to the present invention, the rising temperature limits the gas available due to reduced solubility and reduces the effects. However, the reduction in this effect is offset by an increase in polarity as the temperature increases. Thus, at temperatures of about 30 ° C or higher, the greater polarity of the gases and gas mixtures affects the action of the gases and gas mixtures more than the solubility properties.

Generally, when the effect on the enzyme is increased, the molar concentration of the noble gas or noble gas mixtures is raised above the standard temperature and pressure (STP). In addition, the effects affect the basis for the interaction of gas molecules with enzymes that are independent but can be enhanced or made possible by a mixture of other gases. Note that mixtures of noble gases behave similarly to pure noble gases and effects of mixtures of several gases are found to be similar effects observed with a single noble gas.

25th Furthermore, according to the present invention, it has been found that a specific noble gas or mixtures thereof can inhibit or enhance the enzymatic effect as a function of temperature.

Furthermore, according to the present invention, it has been found that, at generally suitable pH, temperature, pressure, [E] and [C], all enzymes can be specifically inhibited by each of the noble gases of the present invention.

More specifically, the invention relates to a method for improving enzyme activity, wherein at least one enzyme selected from the group consisting of hydrolases, lyases, isomerases and ligases is reacted with pH, temperature, pressure, enzyme concentration (E) and substrate (E). ) with the substrate (s) in which, for at least a part of the reaction time, the enzyme is in contact with one or more noble gases and the noble gases used are neon, argon, krypton or xenon.

The invention further relates to a method for controlling enzyme activity in a reaction between an enzyme for 10 min and its substrate in a liquid medium, wherein the change from an inhibitor of enzyme activity to an enzyme activity enhancer and vice versa occurs with selected from the group consisting of oxidoreductases, lyases, isomerases, and ligases, is reacted in a liquid medium with its substrate, during which at least a portion of the atmosphere surrounding the liquid medium is replaced by one or more noble gases, such as neon, argon, krypton or k is between liquid nitrogen temperature of-120 ° C and 1Cf.sub.s.

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The present invention can be used to control enzyme effects in any application where enzymes are used. For example, the present invention may be used in the advanced production of antibiotics such as penicillin; ethanol and acetic acid production; manufacture of diagnostic instruments; fermentation products such as beer, wine and cheese; and, in general, large-scale enzyme reactions.

As noted above, the present invention can be used successfully to control the effects of enzymes from any of the six identified enzyme categories. The following examples are illustrative and are not intended to limit the invention.

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In particular, the present invention can be used with great success in the regulation of oxidoreductases including, for example, dehydrogenases, oxidases, peroxidases, hydroxylases and oxygenases. Specific examples oksidoreduk-taaseista are tyrosinase, glucose oxidase, asetoiinidehydrogenaasi, tauriinide 5-dehydrogenase, oktopiinidehydrogenaasi, atsobentseenireduktaasi, asetoindoksyy- oxidase, hypotauriinidehydrogenaasi, pseudomonas cytochrome oxidase, 3-hydroxy-siantranilaattioksidaasi, chloride peroxidase, cytochrome C3 hydrogenase, meliotaatti 3-mono-oxygenase, CDP-4-keto-6-deoxydeglucose reductase and rubredoxin NaD + reductase and chlorate reductase, for example. However, any oxidoreductase enzyme 10 may be used.

Another category of enzymes that can be regulated according to the present invention are transferases. Specific examples of transferases include chamosin N-methyl transferase, 3-oxoacylacyl-carrier protein synthase, laminaribose phosphorylase, galactose-6-sulfurylase, diiodothyrosine aminotransferase, sedoheptyl kinase, and lycosin sulfurase transferase, for example. However, any transferase can be used.

A third category of enzymes that can be regulated according to the present invention are hydrolases such as esterases, photopathases, glycosidases and peptidases, for example.

Specific examples of hydrolases include dihydrocoumarin hydrolase, β-D-Glu cosidase, α-glucosidase, ribosome cysteine, acylmuramylalanine carboxypeptidase, ureidosuccinase, floretin hydrolase and 2-haloacid dehalogenase, for example.

However, any hydrolase can be used.

A fourth category of enzymes that can be regulated according to the present invention are lyases, including decarboxylates, aldolases and dehydratases, for example.

Specific examples of lyases include phenylamine ammonium lyase, citrate synthase, methylglyoxyl synthase, ureidoglycolate lyase, allyl lyase, chorismate synthase, and alkylmercury lyase, for example. However, any lyase can be used.

A fifth category of enzymes that can be regulated in accordance with the present invention are isomerases including racemases, epimerases, cis-transisomerases, intracellular oxidoreductases, and intracellular transferases, for example. Specific examples that may be mentioned are glucose phosphate isomerase, UDP arabinose-4-epimerase, maleylacetoacetate isomerase, chorismate mutase, and muconate cycloisomerase, for example. However, any isomerases may be used.

A sixth category of enzymes that can be regulated in accordance with the present invention are ligases which include amino acid RNA ligases, acid thiol ligases, amine synthetases, peptide synthetases and cyclic ligases, for example. Particular mention may be made of acetylcholine synthase, ceryl-2-RNA synthetase, camosine synthetase and methylcarbonyl CoA carboxylase, for example. However, any ligase can be used.

While the present invention generally provides a method for controlling enzyme effects, it also provides a number of specific additional methods.

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First, the present invention provides a method of improving enzyme activity by contacting one or more enzymes with a gas containing one or more noble gases or mixtures thereof.

Second, the present invention also provides a method of inhibiting enzyme effects by contacting one or more enzymes with a gas containing one or more noble gases or mixtures thereof.

Third, the present invention provides a method of altering the optimal pH or / or temperature of one or more enzymes by contacting one or more enzymes with a gas containing one or more noble gases or mixtures thereof.

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Fourth, the present invention provides a specific method for controlling the effects of oxidoreductase enzyme by contacting one or more oxidoreductase enzymes with a gas containing one or more noble gases or mixtures thereof.

5

Fifthly, the present invention also provides a method of specifically regulating the hydrolase enzyme activity by contacting one or more hydrolase enzymes with a gas containing one or more noble gases or mixtures thereof.

10

Sixthly, the present invention also provides a method for specifically regulating the activity of a lyase enzyme by contacting one or more lyase enzymes with a gas containing one or more noble gases or mixtures thereof.

Seventhly, the present invention also provides a method of specifically regulating an isomerase enzyme effect by contacting one or more isomerase enzymes with a gas containing one or more noble gases or mixtures thereof.

Eighth, the present invention also provides a method of specifically regulating the activity of a ligase enzyme by contacting one or more lyrase enzymes with a gas containing one or more noble gases or mixtures thereof.

Ninthly, the present invention also provides a method for specifically regulating a transferase enzyme effect by contacting one or more transferase enzymes with a gas containing one or more noble gases or mixtures thereof.

Tenth, the present invention also provides a method of altering the optimal relative enzyme substrate concentration by contacting one or more of 10,103,884 enzymes with a gas containing one or more noble gases or mixtures thereof.

Nineteenth, the present invention also provides a method for selectively controlling one enzyme in a mixture of two or more enzymes.

It is important to note that the examples of enzymes listed above for each of the six general categories of enzymes are illustrative only and are not intended to limit the invention. According to the present invention, any enzyme 10 can be adjusted using these gases and gas mixtures. For example, just the enzymes used by M. Dixon and E.C. Webb described in Enzymes, Third Edition (Academic Press) can be adjusted according to the present invention.

Generally, as noted above, a gas containing one or more inert gases may be a single pure inert gas or a mixture of inert gases. The gas may also be a mixture of one or more noble gases with other gases, as described above.

The enzymes can be in any form. For example, the adjusted enzyme may be in aqueous, aqueous or organic solution. The enzyme may also be in a different matrix, such as a gel. The enzyme may also be in unbound or bound form and also in bound form in cells, or in living cells such as plants or other tissues.

For example, when the present invention is used to regulate bound enzymes. us, the present invention can be used in numerous applications. For example, bound enzymes can be used in batch reactors, continuous-flow stirred tank reactors, column reactors such as packed bed reactors or even fluid bed reactors.

In addition, the use of bound enzymes is advantageous in cells where the cost and purification of the enzyme extraction is obstructed or when the enzyme is unstable when removed from the natural environment. For example, L-citrulline may be prepared using immobilized cells of Pseudomonas putida and urocanic acid may be prepared using immobilized cells of Achromobacter liquidium.

In addition, the present invention can be used to regulate the effect of an enzyme on enzyme electrodes, such as an electrode used to measure glucose using glucose oxidase.

The present invention can be progressively used whenever it is desired to regulate 10 enzyme effects in a controllable and predictable manner.

To further illustrate the present invention, each of the six basic classes of enzymes will now be described in more detail.

15 I. Oxidoreductase

In general, noble gases strongly inhibit oxidoreductase enzymes. However, the amount of inhibinin varies from one noble gas to another and from one noble gas mixture to another.

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Xenon has the greatest effect on the rate of oxidoreductase reactions and reduces the final equilibrium of the v reactions. All other noble gases inhibit the rate of the reaction less and less impair the final equilibrium of the reaction, depending on their solubility and molecular properties. Krypton has a slightly lower activity than xenon 25 and argon is very active at low temperatures. Thus, according to the present invention. different gas mixtures can be used to optimize the action of oxidoreductase depending on whether ambient or chilled temperatures are used.

By using one or more noble gases or mixtures thereof, various inhibitions of oxidase-ductase are obtained, depending on the exact gas or mixture, the substrate, the temperature and the gas pressure used.

103984 20 Π. transferases

In general, transferase enzymes are generally inhibited. The noble gases or mixtures thereof inhibit, for example, aminotransferase and transpeptidases.

5

In general, krypton has the greatest inhibitory effect, which varies slightly depending on the class of transferase to which it is targeted. In contrast, neon has both the lowest inhibitory and healing activity, depending on the subclass of transferase used.

10 ΠΙ. hydrolases

Generally speaking, noble gases or mixtures thereof have a strong effect on hydrolases.

In particular, xenon has the highest healing effect, while krypton has the lowest healing effect.

However, if desired, the hydrolases can also be inhibited.

20 IV. lyases

In general, noble gases or mixtures thereof provide a strong or moderate enhancement of lysase enzymes. However, some gases inhibit the enzymatic activity of lyase under sub-optimal conditions.

25

Xenon has the greatest contribution, while argon has the least.

V. Isomerases

S

30 1 In general, noble gases or their mixtures, either strongly or moderately, contribute to the isomerase enzymes.

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However, argon and mixtures containing argon may begin to inhibit the enzyme action at higher temperatures.

VI. Ligases (Synthetases) 5

In general, noble gases or their mixtures strongly promote ligase enzymes.

In order to elucidate the procedures used to measure the effect of one or more noble gases on the enzyme in accordance with the present invention, the following representative test protocol description is given by way of example:

General job description:

Solution Preparation: Optimized w / v solutions are prepared by diluting enzyme 15 (units / ml) and substrate (µg / ml) in an appropriate buffer (optimized pH and molarity for enzymes). The solutions are used for gas tests immediately to avoid loss of effect. Various enzymes and substrate concentrations and inhibitors may be used. Physical parameters are varied according to requirements.

20 Spectrophotometric Equipment: Test runs are performed on a Perkin-Elmer Lambda 6 UV / VIS spectrophotometer, temperature controlled, connected to an IBM PS / 2 30 PC (micron). The IMB is loaded with two software products (PECSS or UVDM for recording and studying spectra, modified ENZFITTER and Grafit for kinetic studies).

25th Full Range Spectra: Taking the full range spectrum of the enzyme and substrate species, as well as complete reaction mixtures, allows determination of the appropriate wavelength to follow the enzyme reaction (wavelength corresponding to the main absorbance peak) based on real time. Absorbent species shall be measured and voided. A variety of chromogenic substrates can be used and, under certain conditions, the chromogenic reactions can be coupled to the enzyme reaction being investigated.

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Dilution Ages: Dilution dilutions are run to find the optimal enzyme / substrate ratio to promote gas assays. Reactions are run under optimal conditions, then in super- and sub-optimal substrate regions, then with various inhibitors.

4 5 Preparation of silicone sealed cuvettes: 1 cm light transmitting acrylic or quartz cuvettes are sealed with a silicone rubber closure. The silicone is allowed to cure for 48 hours to obtain gas-tight cuvettes. The cuvettes are blown air to remove any chemical gas impurities as measured by GC / MS and tested for leakage properties.

Preparation of samples: Fill the cuvettes with 2 ml solvent substrate using a gas-tight syringe. The gas-tight serum vials are filled with enzyme and solvent.

Sufficient gas is sequentially bubbled into cuvettes and serum vials with a waiting time of 1 hour between syringes to maximize gas balance. All vials 15 and dead spaces should be flushed with a suitable gas prior to filling. The amount of gas used is measured as sufficient to saturate the required solution.

Comparisons: All possible interfering parameters are controlled including T, P, other gases, air leaks, materials, gas and reagent quality variation, pH.

20 Performing backups.

: ·

Spectrophotometric time run, reaction rate, and kinetic analyzes: Cuvettes and serum vials are gas-impregnated for 40 minutes before running. A gas filled syringe is used to replace the 0.5 ml enzyme solution to avoid air contact. Syringe injections of 0.5 ml of enzyme are made simultaneously to provide a common start time for the samples. 7 gases (air, O2, N2, Ne, Ar, Kr, Xe) are run, each at different * temperatures (0-60 °). Changes in reaction rate or final equilibrium to ambient air are recorded. The results are also compared with oxygen-depleted and de-oxygenated samples. Some samples are also prepared under pressure.

103984 23 Complete Assay Protocol In a complete assay, the enzyme and buffer saline are reacted at optimal pH with five substrate concentrations in solution with noble gas at 5 atm. (saturated solution) at 10% and 50% saturation and pressures of 1.5 and 2 atm. and at least one much higher pressure and auxiliary oxygen equivalents of 10%, 20% and 50% of the total gas at 1 and 2 atmospheric pressures at each temperature in the range 0-65 ° C at 5 ° intervals using N2, 02, Ar, Ne, Kr, Xe, air, and optionally other gases, and by using the desilium mixtures of the above gases, plus the enzyme, are subjected to very high pressures in the hydrostatic control. The controls are also run after evacuation of the solution under vacuum. The reaction is performed colorimetrically with a scanning spectrophotometer in real time and the signals are processed mathematically to obtain differences in speed and yield. The sample numbers are constant and sufficient to ensure correct results. All 15 parameters are independently controlled and measured, and more complex experiments are performed randomly.

The data is obtained in the form of real-time product formation spots (standard velocity curves). Each test cuvette yields one that is patched by the standard method to obtain 20 patches for each of the 6 to 12 cuvette experiments. This patch is attached. We also produce x-y datum tables that form these curves as well as all machine parameters. This data can be further transmitted to calculate differences between curves, yields, rates, and other logical comparisons. These are calculated using a number of discrete program products using simple or complex standard mathematical expressions, linear and non-linear regression fitting, normal logarithmic shifts, enzyme rate calculations (Michaelis-Menten, Eadie, Burk) and time-dependent multi-color earnings analyzes.

To illustrate the present invention more fully, reference will now be made to certain examples, which are provided for purposes of illustration only and are not intended to limit the invention.

24 103984

Class I. Oxidoreductases (ECI)

Example 1 5 Tyrosinase; at 25 ° C and optimum reaction conditions, simple gas saturation of the solution: inert gas or mixture effect

Xe -73% (inhibition) 10 Kr -73%

Ar -60%

No -46.7% 90:10 Xe: Kr ratio -50%

Ar.Xe 99: 1 -70% 15

Example 2

Glucose oxidase: 20 noble gas or mixture effect

Xe -91.6% (inhibition)

Kr -92.7%

Ar -85.8%

Ne -61.7% 25 Category Max:

Xe -95% (inhibition)

£ -91%

Ar -91%

Not -85% 30

It should be noted that the above results depend on the temperature and substrate concentration.

•: x 103984 25

Class II. Transferases (EC2)

Example 3 Gammagglutamyltranspeptidase: noble gas or mixture effect

Xe -7% (inhibition)

Kr -8% 10 Ar -5%

Not -3%

Example 4 Aspartate Aminotransferase: Effect of noble gas or mixture

Xe -17% (inhibition)

Kr -82% 20 Ar -17%

Not -12%

Class III. Hydrolases (EC3) 25

Example 5 26 103984

Beta-D-glucosidase: 5 noble gas or mixture effect

Xe + 40% (improvement)

£ + 14%

Do +16% 90:10 Xe: Kr relation +18% 10

The above results depend on temperature, substrate concentration and substrate type. Addition of various competing substrates resulted in up to 200% improvements using xenon.

15 Class IV. Lyas (EC4)

Example 6

The results vary with temperature and [E / S]. The following values were obtained for the citrate synthetase complex-20 reaction:

15 ° C 25 ° C 10 ° C

Xe +32 0 +18

Kr +32 +6 +37 25 90:10 0 +16 -32

Ar -15 -10 +25

No -14 + 9 + 11 N2 -17 -25 -6 30

Example 7 27 103984

The following values were obtained for the optimized enzyme concentration from phenylalanine ammonia lyase: 5

Xe +18 +3 +5

Kr +7 +4 +4 90:10 +5 +2 +1

Is +6 +1 +3 10 No -2 +6 -6 N2 +19 0 +6

Example 8 The following values were obtained for phenylalanine ammonia lyase when enzyme cong. were below optimum:

Xe +14 +8 +11

Kr +3 +18 +14 20 90:10 +5 +8 +6, Ar -1 +1 +6.5

Ne +15 0 +6 N2 0 0 +12 25 Class V. Isomerases (EC5)

Example 9 28 103984

Triosephosphate isomerase, at 10 ° C: 5 noble gas or mixture effect

Xe +24% (improvement)

£ + 12%

Ar + 8% (but inhibition at -37 ° C at 25 ° C

90:10 Xe: Kr +6.3% 10

Example 10

Phosphoglucose isomerase 15 inert gas or mixture effect

Xe +186% (-61 ° «not learning.)

Kr +206.4%

Ar + 232.5 °

Not +107% (-45 ° Ci opt.) 20. . Marking non-opt. means that the substrate concentration or temperature was not optimal.

Class VI. Ligases (Synthetases) (EC6) 25 1 These enzymes heal, but vary widely; very specific to active | 'to the scene.

Maximum observed improvement in maximum inhibition observed (temperature dependent): "jm

Example 11 29 103984

Acetyl S-CoA Synthetase 5 In a Coupled Complex Reaction Sequence Containing Hydrolytic Enzymes:

Xe + 18.3% / -25.0%

Kr +16.1% / -34.6%

Ar +67.7% / +34.6% 10 Ne +2.3% / -21.9% 90/10 +16.1% / -38.5% N2 +31.2% / - 39.5 %

Example 12 15

As an isolated reaction:

Xe + 15.4% / - 39.5%

Kr +5.0% / -52.6% 20 Ar +75.4% / -27.6%

Those +5.0% / -57.9% 90/10 +35.7% / -118.7% N2 +31.2% / -39.5% In general, inhibition occurs at higher temperatures. Lower temperatures are improving. Nitrogen generally has a much smaller effect than noble gases except at superoptic temperatures. We see noble gases improve this reaction towards optimum yields and velocities under conditions that otherwise would not be optimal for the reaction. We see blend results under optimal conditions that depend on the gas used.

103984 30

The present invention will now be further described with reference to additional examples, which are provided to illustrate the invention and are not intended to limit the invention.

Tyrosinase catalyzed reaction:

Tyrosinase (monophenol, dihydroxyphenylalanine: oxygen oxidoreductase; EC 1.14.18.1) is a monophenol monoxygenase that catalyzes the reaction of orthodiphenols to orthoquinones.

10 Tyrosinase is an important factor in the tanning of fruit and the spoilage of food products.

Test Protocol 15 1. Preparation of solution: 10% w / v solutions are prepared by diluting enzyme (units / ml) and substrate (g / ml) in sodium phosphate nebulizers (pH 6.85, optimized for pH enzyme). The solutions are stored in a refrigerator (0-5 ° C) and used for gas tests for two to three days to avoid loss of effect.

20

2. Spectrophotometric equipment: Test runs were performed on Perkin-Elmer Lambda 6 UV / VIS

'With a spectrophotometer attached to an IBM PS / 2 30 PC (micron). IBM is loaded with two software products (PECSS for spectrum registration and viewing, ENZFITTER for kinetic studies).

25 3. Full-Range Spectra: Take the full-range spectrum of E, S, and E + S- «! lists a suitable wavelength for monitoring the enzymatic reaction (at a wavelength corresponding to the main absorbance peak).

30 4. Dilution Formulas: Dilution Formulas E versus S, and S Versus E are run to find the optimal [E] / [S] ratio for gas tests. According to the absorbance readings, the best of the two pins (buffer + E or buffer + S) are selected.

103984 31 5. Preparation of silicone sealed cuvettes: The 1 cm light passage acrylic cuvettes are sealed with a clear silicone rubber stopper. The silicone is allowed to cure for 48 hours to obtain gas-tight cuvettes.

6. Preparation of Run Samples: Fill the acrylic cuvettes with 2 ml of substrate solution using a gas-tight syringe. Gas-tight serum vials are filled with enzyme. The 3 x 10 cm gas is bubbled sequentially into cuvettes and serum and vials at one hour injection intervals. This represents 10 times the dead volume replacement while maximizing gas release. All syringes and 10 dead compartments shall be cleaned before filling with suitable gas. After the third 10 cc syringe, the cuvettes and the vials of serum are left overnight with two 10 cc syringes filled with suitable gas in the refrigerator at 0-5 ° C.

15 7. Comparisons: All possible interfering parameters are monitored including T, P, other gas, air leaks, materials, gas and reagent quality variability, pH. Relevant repetitions will be performed.

8. Spectrophotometer runs: Fill the cuvettes and serum vials with 10 cubic centimeters to 20 meters of gas for 40 minutes before running. A gas-filled syringe is used. To withhold 0.5 ml enzyme solution to avoid air contact. Syringe 0.5 ml syringes of enzyme are run concurrently to provide a common start time for tQ samples. The seven gases (air, O 2, N 2, Ne, Ar, Kr, Xe) are each run at five temperatures (15 ° C, 20 ° C, 25 ° C, 30 ° C, 35 ° C). Changes in the velocity of the reaction and the surrounding air or the final equilibrium with the surrounding air are recorded.

103984 32 2. REAGENTS:

Tyrosinase (Sigma Chemical Co., St Louis, MO); 5 - Catalog # T-7755 (Lot 48F-9610) - Monophenol monooxygenase; polyphenol oxidase; catechol oxidase; mono-phenol, dihydroxyphenylalanine; oxygen oxidoreductase; EC 1.14.18.1) 10 - sponge - 25000 units 12 mg solids 2200 units / mg solids (tyrosinase effect) - Tyrosinase unit assay: one unit causes an increase of 0.001 per minute at 15 ° C at pH 6.5 C, using L-tyrosine as a substrate. Reaction volume 3 ml (1 cm light passage)

- Store in a dry place at temperatures below 0 ° C

20 L-tyrosine (Sigma Chemical Co., St Louis, MO); - Catalog No. T-7755 (Lot 48F-9610) - L-3- [4-Hydroxyphenyl] alanine - Free Base (pfs) Crystalline 25 - Anhydrous Mol. towards. 181.2 - Store at room temperature (25 ° C)

Sodium Phosphate Monobasic (E K Industries, Addison, IL) - Catalog No. 8680 30 - NaH2 P04 * H20 - Reagent Crystals - FW 137.99 103984 33

Sodium Phosphate Dual Alkaline (E K Industries, Addison, IL) - Catalog No. 8720 - Na2 HP04 5 - Anhydrous - FW 141.96

Deionized water H2O (Bamstead NANOpure II) 10 3. PREPARATION OF THE SOLUTION:

Sodium phosphate buffer pH 6.85 (25 ° C) - 138 g NaH 2 PO 4 * H 2 O 15 - 142 g Na 2 in HPO 4 - 20 L D.I. H 2 O - stored at room temperature (25 °) in a plastic acid bottle Tyrosinase solution (100 µg / ml; 208 units / ml; 2.08 units / µg): 20-10% w / v Na in phosphate buffer * - mixed by inversion several times to dissolve the contents - stored in a refrigerator (0-5 ° C) in a 125 ml HDPE bottle wrapped in aluminum foil (to prevent light dependent decomposition) 25 L-tyrosine solution (100 µg / ml): - 10 w / v Na in phosphate buffer - magnetic stirring (25 ° C, 30 min) 30 - Store in a refrigerator (0-5 ° C) in a 250 ml amber bottle 103984 34 4. GAS ATMOSPHERES:

Air (ambient)

Argon (Alphagaz, Research Quality) 5 Krypton (Alphagaz, Research Quality, Minimum Purity 99.995% [ppm])

Neon (Alphagaz, Research Quality, Minimum Purity 99.999% [ppm])

Nitrogen (Alphagaz, Research Quality, Minimum Purity 99.995% [ppm])

Oxygen (Alphagaz, Research Quality, Minimum Purity 99.997% [ppm])

Xenon (Alphagaz, Research Quality, Minimum Purity 99.995% [ppm]) 10 5. APPARATUS AND MATERIALS: 5.1. Means:

Perkin-Elmer Lambda 6 UV / Vis Spectrophotometer (Narrowband Spectrophoto-15m) equipped with Automatic Five-Cell Thermoelectric Transport Holds (5x5 sample and reference cell holder, Model C 005-0515)

Perkin-Elmer Lambda Accessory Interface (Model C691-0000) 20 Perkin-Elmer Digital Controller (Model C 570-0701)

Perkin-Elmer Temperature Programmer (Model C 570-0710) IBM PS / 2 Micro 25 Epson EX800 Printer Software: - IBM DOS (Disk Drive System Version 3.30, Boca Raton, FL) - PECSS (Perkin-Elmer Computer Spectroscopy Software) , Norwalk, CT) 30 - ENZFITTER (Nonlinear Regression Data Analysis Software, Elsevier-BIOSOFT,

Cambridge, UK) - PIZAZZ PLUS (Print Enhancement Software, APTEC, Pepperell, MA) 103984 35

Mettler ΑΕΙ00 equilibrium: - weight range: 0-109 g - readability: 0.1 mg 5 Bamstead NANOpure II cartridged deionization system, Tokyo Rikakikai Co. Micro Tubing Pump MP-3 (Perkin-Elmer Water Cooled Cells) 5.2. Materials: 10 100, 200 ml bottles of FISHERbrand acrylic cuvettes (standard type / methacrylate / UV grade / disposable): 1 cm light flow, square cuvettes are capable of holding 3 ml solution in 100% silicone rubber closure (clear) with 3 ml rubber sealed serum vials 15 1 ml 1/100 ml disposable pipettes 1 cm 1/100 cm disposable tuberculin syringes

T O

10 cm 1/5 cm disposable syringes 20GJ '/ 2 disposable needles 20 6. TECHNICAL DEVELOPMENT: 6.1. Initial observations: Full-range runs (900-190 nm) were run with tyrosinase, L-tyrosine and the final products of the enzyme reaction.

25 6.1.1. Experimental setup: PARAM: absorbance gap 1 nm 30 rpm 1600 nm / minute response time 1 second auto-recording yes 36 103984 AZERO: background correction (900-190 nm) SCAN: data interval 1.0 nm Temperature: ambient (26 ° C), temperature programming off top 5 6.1.2 Full range driving:

(900-190 nm) Tyrosinase Run (Fig. 1): sample = 2.5 ml tyrosinase (100 / ml) blind = 2.5 ml sodium phosphate buffer 10 Filing Name: T775501.SP

(900-190 nm) run of tyrosinase (Fig. 2): sample = 2.5 ml tyrosinase (100 μ ^ νολ) blind = 2.5 ml of sodium phosphate buffer Filing name: T375401.SP 15 (900-190 nm) run of final products ( Fig. 3-4): sample = 2 ml L-tyrosinase (100 µg / ml) + 0.5 ml tyrosinase (100 µg / ml) for 20 minutes after reaction blind = 2 ml L-tyrosinase (100 µg / ml) ml) 20 + 0.5 ml Na phosphate buffer

Archive name: TCEB0081.SP

6.1.3. Determination of the main absorbance peaks: 25 A graphical cursor (home key) was used for this purpose.

»J

Tyrosinase: Absorbance peak at 275 nm (Protein) L-Tyrosine: Absorbance peak at 275 nm

Reaction end products: absorbance peak at 480 nm and 305 nm.

30 103984 37 6.1.4. Determination of the appropriate wavelength to follow the reaction:

Examination of three full-range spectra (Fig. 5) suggests that the enzymatic oxidation reduction of L-tyrosine is optimally observed at either 480 nm or 305 nm.

6.1.5. Determination of an appropriate [E] / [S] ratio: T-3754 T-7755 Na Phosphate Final 10 (100 / ml / ml) (100 / ml / ml) Buffer [E] (ml) (ml) (ml) (100 Mg / ml) Sample 5 2 0.1 0.4 20 Sample 4 2 0.2 0.3 40 15 Sample 3 2 0.3 0.2 60 Sample 2 2 0.4 0.1 80 Sample 1 2 0.5 0.0 100

Time runs were executed using the Cell Programming (CPRG) command, which stores 20 time run data simultaneously from up to 5 cells.

Wavelength 480 nm: run (Fig. 6) 45 points, 6 second intervals => 45 min run 25 TCEB0091.SP [tent] = 100 µg / ml cell 1 TCEB0092.SP [tent] = 80 µg / ml cell 2 TCEB0093.SP [tent] = 60 Mg / ml cell 3 TCEB0094.SP [tent] = 40 ug / ml cell 4 30 Wavelength 305 nm: run (Fig. 7) 60 points, at 60 second intervals == => 1 hour run 103984 38 TCEB0101.SP [enzymes] = 100 µg / ml cell 1 TCEB0102.SP [enzymes] = 80 µg / ml cell 2 TCEB0103.SP [enzymes] = 60 µg / ml cell 3 TCEB0104.SP [enzyme] = 40 µg / ml cell 4 5 TCEB0105.SP [enzyme] = 20 pg / ml cell 5

Conclusion:

The optimum conditions are [E] / [S] (100 pg / ml) / (100 μg ml), the absorbance changes are observed at 305 nm.

6.2. Preliminary tests: 6.2.1. Preparation of samples and comparisons:

Xenon impregnated samples (see sampling procedure)

Air samples: Acrylic cuvettes filled with 2 ml of substrate T-3754 with a 1 ml pipette and covered with a plastic cap. This procedure was later changed as the transition from plastic hoods to silicone sealed caps was introduced. No significant difference was observed.

Comparison (blinds): acrylic cuvettes covered with a plastic dome.

- B1: 2 ml T-3754 (100 pg / ml) + 0.5 Na phosphate buffer 25 - B2: 2 ml Na phosphate buffer + 0.5 ml T-7755 (100 mg / ml) 6.2.2. Time travel at 305 nm:

Blind Nl: 30

- AIR sample: filename = TCEB0114.SP

- Xenon sample: filename = TCEB0115.SP

39 103984

Blind B2:

- AIR sample: filename = TCEB0116.SP

- Xenon sample: filename = TCEB0117.SP 5 L-tyrosine monoclonal (B1) was found to cause absorbance readings, so the tyrosinase labeled assay (B2) was preferred.

These 4 time runs show the optimum inhibition curve for xenon. In subsequent 10 runs (6.3.7. And 7.2.), The L-tyrosine solution is contaminated with time and causes a lower absorbance reading.

6.3. Technological development: 15 6.3.1. Step 1: Production of silicone sealed cuvettes

The acrylic disposable cuvettes are closed with a clear silicone rubber closure. Allow the silicone to dry for 24 hours. After 24 hours, we have gas-tight acrylic cuvettes. This was tested by bubbling under water.

20 6.3.2. Step 2: Substrate Sampling Procedure 30 40 103984 6.3.3. Step 3: Enzyme Sampling Procedure

Serum vials are filled with 2 ml of enzyme using a 1 ml pipette (1/100 ml) sealed with a rubber septum and pressed with an aluminum cap.

5 Serum vials are gas-tight.

The filled serum vials are stored in a refrigerator (0-5 ° C).

6.3.4. Step 4: Gas Capture Procedure (ARGON, KRYPTON, NEON, OXY 10 AND XENON) 3 x 10 cm gas is sequentially bubbled into silicone sealed cuvettes and serum vials (T-3754, T-7755) every 1 hour between syringes. This corresponds to a displacement of 10 times the dead volume while maximizing gas release.

All syringes and dead areas should be cleaned with suitable gas before filling. After the third 10 cm syringe, silicone-sealed cuvettes and serum vials

O

leave overnight with two 10 cm syringes filled with suitable gas in 20 refrigerator at 0-5 ° C.

6.3. Note:

Air samples (both enzyme and substrate) do not bubble. The acrylic cuvette is sealed with 25 ordinary vinyl hoods (non-gas tight). This was later transferred to silicone ; for use in closed cuvettes. Several comparisons with the concentrated air bubbling method do not show any difference between the two methods.

6.3.6. Step 5: Spectrophotometric runs a. Silicone sealed cuvettes, serum vials and plastic sealed cuvettes are removed from the refrigerator. This procedure will be changed later (see 7.1.5.) n syringe.

c. The cuvettes are placed in the cell holder and allowed to equilibrate at a temperature corresponding to the cell holder temperature for 10 minutes.

d. The 1 cm syringe is filled with a suitable gas and used to remove 0.5 ml of the solution from the vial of serum, this is to avoid the introduction of air into the vial during the enzyme sample.

10 e. Driving:

Sequential syringes of enzyme 0.5 ml into cuvettes are performed as quickly as possible from cell 5 to cell 1 (residence time x = 2 sec). This method was later switched to simultaneous injection.

15 6.3.7. Spectrophotometric runs: Time runs at 305 nm 60 dots, 60 second intervals => 1 hour run 20 Run 1: Tl = 15 ° C (Tg = TR = 15.1 ° C)

Filing Name gas cell no B1TIG2.SP neon cell 2 25 B1TIG3.SP argon cell 3 B1TIG4.SP krypton cell 4 B1TIG5.SP xenon cell 5

Run 2: Tl = 15 ° C (Tg = TR = 15.1 ° C) 42 103984

Filing Name Gas Cell # 5 B1TIG6.SP Air Cell 1 B1TIG7.SP Oxygen Cell 2

Run 3: Tl = 20 ° C (Tg = TR = 20 ° C) 10 Filing Name Gas Cell # B1T2G1.SP Air Cell 1 B1T2G2.SP neon cell 2 B1T2G3.SP argon cell 3 15 B1T2G4.SP krypton cell 4 B1T2G5 .SP Xenon Cell 5

Run 4: Tl = 20 ° C (Tg = TR = 20 ° C) 20 Filing Name Gas Cell # «B1T2G6.SP Oxygen Cell 1

Run 5: Tl = 25 ° C (Tg = TR = 24.9 ° C)] 25 | , Filing name for gas cell no! 'B1T3G1.SP air cell 1 B1T3G2.SP neon cell 2 30 B1T3G3.SP argon cell 3 B1T3G4.SP krypton cell 4 B1T3G5.SP xenon cell 5

Run 6: Tl = 25 ° C (Tg = TR = 24.9 ° C) 43 103984

Filing Name Gas Cell # 5 B1T2G6.SP Oxygen Cell 1

Run 7: Tl = 30 ° C (Tg = TR = 29.9 ° C)

Filing Name Gas Cell # 10 _ B1T4G1.SP Air Cell 1 B1T4G2.SP Neon Cell 2 B1T4G3.SP Argon Cell 3 B1T4G4.SP Krypton Cell 4 15 B1T4G5.SP Xenon Cell 5

Run 8: Tl = 30 ° C (Tg = TR = 29.9 ° C)

Filing Name Gas Cell # 20 _ B1T2G6.SP Oxygen Cell 1

Run 9: Tl = 35 ° C (Tg = TR = 34.9 ° C) 25 Filing Name Gas Cell # B1T5G1.SP Air Cell 1 B1T5G2.SP neon cell 2 B1T5G3.SP argon cell 3 30 B1T5G4.SP krypton cell 4 B1T5G5.SP Xenon Cell 5

Run 10: Tl = 35 ° C (Ts = TR = 34.9 ° C) 44 103984

Filing name gas cell # 5 B1T2G6.SP oxygen cell 1 7.3. Graphic results:

Time Drives / Temperature - Connected Pages 10 Time Drives / Gas - Connected Pages 7. FINAL METHOD: 7.1. Final test protocol: 15 7.1.1. Step 1: Production of silicone sealed cuvettes

Same as 6.3.1. but silicone is allowed to cure for 48 hours to remove acetic acid vapors that could affect enzyme inhibition. This is confirmed by analysis.

7.1.2. Step 2: Method of making the substrate

Same as 6.3.2 25

Blank B2 (2 ml Na phosphate buffer + 0.5 ml T-7755) is prepared according to the same procedure (in a silicone sealed cuvette) but does not undergo the gas impregnation step (7.1.4.).

The comparisons show no absorption of gas in the occupational absorption area.

103984 45 7.1.3. Step 3: Enzyme Sampling Method

Same as 6.3.3.

5 7.1.4. Step 4: Gas impregnation method (AIR, ARGON, KRYPTON, NEON,

Nitric Oxygen (Xenon) 3x10 cm gas is bubbled into silicone sealed cuvettes and serum vials (T-3754, T-7755) at 20 minute intervals (at 0.5 ° C) between each gas syringe.

After the third 10 cm syringe, the cuvettes and the serum vials are left overnight under two 10 cm syringes filled with suitable gas in a refrigerator at 0.5 ° C.

15 7.1.5. Step 5: Spectrophotometric runs a. Silicone-sealed cuvettes are removed from the refrigerator 40 minutes prior to test run * 1, filled with 10 cm of gas, and left at room temperature (26 ° C) under two 1 x 10 cm syringes.

Serum vials are removed from the refrigerator 25 minutes prior to test run, filled with 10 3 3 cm of gas and returned to the refrigerator under two 10 cm syringes.

25 c. 15 minutes before driving, the 10 cm syringes (and needles) are removed with silicone; closed cuvettes. The cuvettes are placed in the cell holder and allowed to equilibrate at the same temperature as the cell holder for 10 minutes.

. #. * 3 days 5 minutes before driving, the 1 cm syringe is filled with gas and used to obtain 0.5 ml of the 30 enzyme solution from the serum vials to avoid airflow into the vial while collecting the enzyme.

103584 46 Driving Directions:

Silicone-sealed cuvettes are quickly removed from the cell holder and sealed to prevent gas bubbles that may form as the cuvettes become warm (especially at 30 and 35 ° C) and reinserted in the cell holder.

5 ml aliquots of enzyme are simultaneously run to obtain a total starting time tg for the samples.

7.2. Spectrophotometric runs: time runs at 305 nm at 10 200 dots, 18 second intervals => 1 hour run 7.2.1. Run 1: Tl = 15 ° C (g = TR = 15.1 ° C) 15 Filename gas cell no B2TIG1.SP air cell 1 B2TIG2.SP neon cell 2 B2TIG3.SP argon cell 3 20 B2TIG4.SP krypton cell 4 B2TIG5.SP Xenon Cell 5 7.2.1. Run 2: Tl = 15 ° C (Tg = TR = 15.1 ° C) 25 Filename gas cell no B2TIG6.SP air cell 1 B2TIG7.SP oxygen cell 2 B2TIG8.SP nitrogen cell 3 30 7.2.2. Run 3: Tl - 20 ° C (Tg = TR = 19.9 ° C) 47 103984

Archive name gas cell no 5 B2T2G1.SP air cell 1 B2T2G2.SP neon cell 2 B2T2G3.SP argon cell 3 B2T2G4.SP krypton cell 4 B2T2G5.SP xenon cell 5 10 7.2.4. Run 4: Tl = 20 ° C (Tg = TR = 19.9 ° C)

File name gas cell no 15 B2T2G6.SP air cell 1 B2T2G7.SP oxygen cell 2 B2T2G8.SP nitrogen cell 3 7.2.5. Run 5: Tl = 25 ° C (Tg = TR = 24.9 ° C) 20, Filename gas cell # B2T3G1.SP air cell 1 B2T3G2.SP neon cell 2 25 B2T3G3.SP argon cell 3 B2T3G4.SP krypton cell 4 1 - - B2T3G5.SP xenon cell 5 48 103984 7.2.6. Run 6: Tl = 25 ° C (Tg = TR = 24.9 ° C)

Filename Gas Cell # 5 B2T2G6.SP Air Cell 1 B2T2G7.SP Oxygen Cell 2 B2T2G8.SP Nitrogen Cell 3 7.2.7. Run 7: Tl = 30 ° C (Tg = TR = 29.9 ° C) 10

Archive name gas cell no B2T4G1.SP air cell 1 B2T4G2.SP neon cell 2 15 B2T4G3.SP argon cell 3 B2T4G4.SP krypton cell 4 B2T4G5.SP xenon cell 5 7.2.8. Run 8: Tl = 30 ° C (Tg = TR = 29.9 ° C) 20

Filename Gas Cell No. B2T2G6.SP Air Cell 1 B2T2G7.SP Oxygen Cell 2 25 B2T2G8.SP Nitrogen Cell 3 103984 49 7.2.9. Run 9: Tl = 35 ° C (Tg = TR = 34.9 ° C)

Filename gas cell # 5 B2T5G1.SP air cell 1 B2T5G2.SP neon cell 2 B2T5G3.SP argon cell 3 B2T5G4.SP krypton cell 4 B2T5G5.SP xenon cell 5 10 7.2.10. Run 10: Tl = 35 ° C (Tg = TR = 34.9 ° C)

Filename gas cell # 15 B2T2G6.SP air cell 1 B2T2G7.SP oxygen cell 2 B2T2G7.SP nitrogen cell 3 7.3. Graphic results: 20

Time Drives / Temperature - Connected Pages Time Drives / Gas - Connected Pages

7.4. Enzyme kinetics: ENZFITTER

25

From the above results obtained from the tyrosinase L-tyrosine reaction, the following conclusions can be drawn.

Xenon significantly inhibits and reduces the rate of the final equilibrium of the tyrosinase L-tyrosine reaction. In addition, all other noble gases inhibit the rate of the reaction and reduce the final equilibrium, depending on both their solubility and molecular properties. Krypton generally has the same but lower activity as xenon, while argon is surprisingly active even at low temperatures.

In addition, the effects of oxygen loss can be controlled by the use of nitrogen. Compared to 5 argon, it is found that nitrogen has no physical ability to interact with the active site. It turns out that argon is more active in relation to nitrogen. This also seems to apply to other classes of enzymes.

Because the differences in solubility can only account for some of the inhibitions observed, active site interactions or protein structural changes are bound to occur there.

The kinetic experiments are shown below.

High pressure tests: 15

The tests were carried out in pressure cells pressurized with additional test gas after the solution had already been saturated, in 2 atmospheres or 3 atmospheres in real time or after pressurization in a 1 liter cylinder for 1 or 24 hours at 30.6 atmospheres and 100 atmospheres.

20

The following experiments were used.

Protocol: Effects of high pressure on enzymes.

Theory: We put the enzymes under high pressure and determine their activity. High pressures should inhibit the action of enzymes.

: Enzyme: Tyrosinase (SIGMA No. T-7755) (Monophenol monooxygenase; polyphenol oxidase; catechol oxidase; monophenol, dihydroxyphenylalanine: oxygen oxidoreductase; EC 30 1.14.18.1)

Of mushrooms 103984 51

Definition of Tvrosinase Unit:

One unit results in an increase of 0.001 per minute A2gQ at pH 6.5 at 25 ° C in a 3 mL reaction mixture containing L-tyrosine.

5 Effect of tyrosinase: 3870 U / mg solid 7.1 g solid => 27440 units Stored at below 0 ° C Substrate: L-tyrosine (SIGMA No. T-3754) L-3- [4-Hydroxy-phenyl] ] alanine 10 Free base (pfs) crystalline

Anhydrous Molecule. towards. 181.2 Store at room temperature (25 ° C)

Enzyme: β-D-glucosidase (SIGMA No. G-4511) (Emulsion; β-D-glucosidase glucohydrolase EC 3.2.1.21)

Unit Definition:

One unit releases 1.0 µmol of glucose from salicin per minute at pH 5.0 at 37 ° C.

Effect: 22 U / mg solids 20 to 12 g solids => 264 units

Store dried at 0-5 ° C Lot # 49F-4021

Substrate: p-nitrophenyl-B-D-glucopyranoside (SIGMA No. N-7006)

Crystalline 25 Contains 2.4% solvent

Anhydrous Molecule. towards. 301.3 *

': Store dried at room temperature below 0 ° C

Lot # 129F-5057 Koel: 4/10 / 91-4 / 11/91 30 Gases: 1. Air 2. N2 103984 52

Pressure: 1. 30.6 atm (450 psi) 2. 100.0 atm (1470 psi)

Comparisons: Comparison A: The enzyme was not pressurized or placed in a gas cylinder.

Comparison B: The enzyme was not pressurized or placed in the gas cylinder 5 1. Comparison for gas 1 (air).

Comparison B: The enzyme was not pressurized or placed in the gas cylinder 2. Comparison for gas 2 (N2).

Preparation of the solution: 4/10/91

Solution A: Sodium phosphate buffer pH 6.6 at 25 ° C 10 IL deionized water 141.96 x 0.2 x 187.5 x 1/1000 = 5.3 g Na 2 HPO 4 119.96 x 0.2 x 312.5 x 1/1000 = 7 , 5 g NaH 2 PO 4 pH: 6.502

Solution B: Tyrosinase solution in Na phosphate buffer (228 U / ml) 15 14.2 mg T-7755 diluted to 240 mL Na phosphate buffer pH 6.6

at 25 ° C

Solution C: 50 μg / ml L-tyrosine solution in sodium phosphate buffer 10 mg T-3754 diluted in 200 ml Na phosphate buffer pH 6.6 at 25 ° C.

Solution D: 50 μξ / τηΐ solution of L-tyrosine in sodium phosphate buffer pH 6.8 at 25 ° C: 2 L deionized water 2 x 141.96 x 0.2 x 245 x 1/1000 = 13.91 g of Na 2 HPO 4 2 x 119 , 96 x 0.2 x 255 x 1/1000 = 12.20 g NaH 2 PO 4 pH tested: 6.719

Solution E: 100 μl / ιη1 β-D-glucopyranoside solution in sodium phosphate buffer t 25 mg N-7006 diluted in 250 ml Na phosphate buffer pH 6,8 at 25 ° C.

Solution F: β-D-glucosidase solution in Na phosphate buffer pH 6.8 (25 ° C) (2.18 to 30 units / ml)

24 mg G-4511 diluted in 242 mL Na phosphate buffer pH 6.8 at 25 ° C

103984 53

Method:

Tyrosinase: 4/11/91

Place a 100 ml aliquot of the enzyme solution in each of the two disposable gas cylinders. Shake the enzymes in each cylinder for several minutes to 5 minutes and then remove the 5 ml sample for comparison B and comparison C.

Remove the cylinders to the stub, gasify with a suitable gas; slowly pressurize to 30.6 atm. Leave for 60 minutes at the required pressure. Slowly reduce the pressure to 2 psi before bringing it to the laboratory.

Transfer about 10 mL of enzyme from each cylinder while keeping it in 10 gas in separate containers and run TDrive / CPRG Enzyme / Substrate immediately.

Enzyme 1, Gas 1, Pressure 1, Repetition 1,

Enzyme 1, gas 2, pressure 1, repeat 1

Bring the cylinders back into the sleeve and re-pressurize to 100 atm with a suitable gas. Leave for 60 minutes at the desired pressure. Slowly lower the pressure to 2 psi before bringing it to the laboratory.

Transfer about 10 ml of enzyme from each cylinder while keeping it in gas in separate containers and run TDrive / CPRG enzyme / substrate mixture immediately.

20 Enzyme 1, gas 1, pressure 2, repeat 1,, enzyme 1, gas 2, pressure 2, repeat 1 β-glucosidase:

Rinse cylinders with equal amounts of D.I. ^ O. Then rinse the cylinders with another enzyme solution (glucosidase). Place a 25 ml deficit of the enzyme solution in each of the two available ': - gas cylinders. Shake the enzyme in each cylinder for several minutes and then remove a 5 ml sample for comparison B and comparison C. Remove the cylinders from the mount, allow suitable gas 30 to flow, slowly raise the pressure to 30.6 atm. Allow to be pressurized for 60 minutes. Slowly depressurize to 2 psi before bringing it to the laboratory. Transfer approximately 10 ml of enzyme 54 103984 from each cylinder while keeping it in gas in separate containers and run the TDrive / CPRG of the enzyme substrate mixture immediately.

Enzyme 2, gas 1, pressure 1, repeat 1 5 enzyme 2, gas 2, pressure 1, repeat 1

Bring the cylinders back to the mounting and pressurize again to 100 atmospheres with suitable gas. Leave for 60 minutes at the required pressure. Slowly depressurize to 2 psi before bringing it to the laboratory. Transfer approximately 10 ml of enzyme 10 from each cylinder into separate containers while keeping it in the gas and run immediately on the TDrive / CPRG enzyme / substrate mixture.

Enzyme 2, gas 1, pressure 2, repeat 1 15 Enzyme 2, gas 2, pressure 2, repeat I

Spectrum: 25 ° C

Cuvettes: 34 minimum PARAM: Slot 1 20 speed 1500. , save yes no CPRG: T tyrosinase: 305 nm

25 80 points => 20 min RUN

16s int I Ymin -

Ymax? 2 ° B-glucosidase: 30,400 nm 40 dots ==> 10 min RUN 16 sec int 103984 55 Y = 0.0 min 'Υ = 15 max

Blinds: Tyrosinase: 2 mL solution A + 0.5 mL solution B

β-glucosidase: 2 ml solution E + 0.5 ml solution D

Sample: Tyrosinase: 2 ml solution C + 0.5 ml solution B

β-glucosidase: 2 ml solution E + 0.5 ml solution F

Cell Conveyor: Cell 1: Comparison A

Cell 2: Comparison B Cell 3: Comparison C

10 Cell 4: Gas 1 (Air)

Cell 5: Gas 2 (N2)

Archives: Tyrosinase: X1P1G1C1 ... 5.SP => X1P1G1C1 ... 5.SP was renamed X1P1E1C1 ... 5.SP thru DOS.

15 X1P2E 1C 1 ... 5.SP => ... 5.SP started with higher absorbance, so we let out gas bubbles and did a full range run for all five cells. Full range travel 900-190 nm. Archiving: X1SCAN1 ... 5.SP.

20 10 ARCHIVING: β-glucosidase:

X2P1E2C1 ... 5.SP

X2P2E2C1 ... 5.SP

25 10 ARCHIVING:

Experiment 2: 4/15/91 In this experiment, we only perform tyrosinase pressure tests.

Gases: 1. Air 30 2. N2

Pressures: 1. 30.6 atm (450 psi) 2. 100.0 atm (1470 psi) 103984 56

Comparisons: Comparison A: The enzyme was not pressurized or placed in a gas cylinder.

Comparison B: The enzyme was not pressurized or placed in the season cylinder 1. Comparison for gas 1 (air).

Comparison B: The enzyme was not pressurized or placed in the gas cylinder 5 2. Comparison for gas 2 (Ν2).

Preparation of the solution: 4/10/91

Solution A: Sodium phosphate buffer pH 6.6 at 25 ° C 1 L deionized water 141.96 x 0.2 x 187.5 x 1/1000 = 5.3 g Na 2 HPO 4 10 119.96 x 0.2 x 312.5 x 1/1000 = 7.5 g NaH 2 PO 4 pH tested: 6.502

Solution B: Tyrosinase solution in Na phosphate buffer (228 U / ml)

14.2 mg T-7755 diluted in 240 mL Na phosphate buffer pH 6.6 at 25 ° C

Solution C: 50 L solution of tyrosine in sodium phosphate buffer 10 mg T-3754 diluted in 200 ml Na phosphate buffer pH 6.6 at 25 ° C.

Method: Tyrosinase: 20 Place a 25 ml aliquot of the enzyme solution in each of the two disposable gas cylinders. Shake the enzyme in each cylinder for several minutes and then remove 5 ml of sample for comparison B and comparison C. Remove the cylinders to the stub, gasify with a suitable gas. Pressurize and depressurise the gas cylinders several times to remove the main space / Tail gas. Slowly raise the pressure to 30.6 atm. Anna. be at the desired pressure for 60 minutes. Slowly depressurize to 40 psi before bringing it to the laboratory. Transfer about 5 ml of the enzyme from each cylinder while keeping it gas in separate dishes and run immediately on the TDrive / CPRG enzyme / substrate mixture.

30 Enzyme 1, gas 1, pressure 1, repeat 2

Enzyme 1, gas 2, pressure 1, repeat 2 Enzyme 1, gas 2, pressure 2, repeat 2 103984 57

Spectrum: 25 ° C

Cuvettes: 10 min PARAM: Slot 1 speed 1500 5 save yes no 'CPRG: Tyrosinase: 305 nm 80 dots ==> 20 min RUN 10 16s int ^ min ~ ^ max

Blinds: Tyrosinase: 2 mL solution A + 0.5 mL solution B

Sample: Tyrosinase: 2 ml solution C + 0.5 ml solution B

15

Tests to determine if solution C was still useful.

TDrive: 305 nm, 80 pts, 16 s int.

S = 2.0 mL C + 0.5 mL B 20 R = 2.0 mL A + 0.5 mL D

Archives: TYTRL1.SP

<

The cell vehicle:

Run 1: 100 atm Cell 1: Comparison A 25 Cell 2: Comparison B

Cell 3: Comparison C

'* Cell 4: gas 1 (air)

Cell 5: Gas 2 (N2) 30 Run 2: 30 atm

Cell 1: Comparison A Cell 2: Comparison B

103984 58

Cell 3: Comparison C Cell 4: Gas 1 (Air)

Cell 5: Gas 2 (N2)

Archives: Tyrosinase:

5 X7P2E1C1 ... 5.SP

X7P1E1C1 ... 5.SP

10 ARCHIVES:

Experiment 3: 4/24/91 (Solution ready and initial pressure), 10 4/25/91, 4/26/91 In this experiment, we only perform tyrosinase pressure tests.

Gases: 1. Air 2.N2 15 3. Xe: 1 atm / N2 pressure

Pressure: 1. 30.6 atm (450 psi) 2. 100.0 atm (1470 psi)

Comparisons: Comparison A: The enzyme was not pressurized or placed in a gas cylinder.

20 Preparation of the solution 4/24/91. Solution A: Sodium phosphate buffer pH 6.8 at 25 ° C: 1 · 2 L deionized water. 12 x 141.96 x 0.2 x 245 x 1/1000 = 13.91 g Na 2 HPO 4 x 119.96 x 0.2 x 255 x 1/1000 = 12.20 g NaH 2 PO 4 pH tested: 6.740

Solution B: Tyrosinase solution in Na phosphate buffer (228 U / ml)

T

14.2 mg T-7755 diluted in 240 ml Na phosphate buffer pH 6.8 at 25 ° C.

Solution C: 50 µg / mL L-tyrosine solution in Na phosphate buffer 30 10 mg T-3754 diluted in 200 mL Na phosphate buffer pH 6.8

mp 25 ° C

103984 59

Method:

Tyrosinase: 4/24/91 Cylinder Preparation: The cylinders (3) were then vacuum-injected with 50 cm3 of D.I. H20 and put under pressure: 5 Cylinder 1 with air

Cylinder 2 with nitrogen N2 Cylinder 3 with nitrogen N2 to 60 psi, shaken, turned up and released to blow fluid out of the cylinders. This procedure was repeated with 3 x 10 µl 50 cm @ 3 of sodium phosphate buffer.

The cylinders were pressurized as described above three times to remove any fluid from the cylinders.

Enzyme Injection: The cylinders were again placed under vacuum and injected with 60 cm of tyrosinase solution using vacuum to suction the enzyme into the cylinder. The cylinders were pressurized as described above.

Cylinder pressure was released and the cylinders were pressurized 10 times to remove C ^ from the cylinders.

Final pressurization: Cylinders 1 (air) and 2 (N2) were pressurized with the respective gas to 30.6 atmospheres. Cylinder 3 was pressurized to 1 to 20 atmospheres with Xe, then pressurized to 30.6 atmospheres to maintain Xe.

Schedule: Cylinders were pressurized to 30 atmospheres at 1.00 i.p. 4/24/91. Due to the use of 25.4.91 spectrophotometers at a temperature of 5 ° C for a different experiment, it was decided to sample the cylinders at the end of the day when the spectrophotometers were available at a constant temperature of 25 ° C. Sampling took place 28 hours after pressurization **. Spectrum / gas run was performed immediately on these samples.

4/25/91 30 Repressurization: The cylinders were pressurized again at 6:00 pm on 4/25/91 to 100 atm. These cylinders are collected at 2:00 pm on 4/26/91 (20 hours after pressurization).

103984 60

Spectrum: 25 ° C

Cuvettes: 10 min PARAM: Slot 1 Speed 1500 5 Save Yes No CPRG: Tyrosinase: 305 nm 80 dots => 20 min RUN 10 16s int

Ymin - ° '°

Ymax = 2 '°

Blinds: Tyrosinase: 2 mL solution A + 0.5 mL solution B

Sample: Tyrosinase: 2 ml solution C + 0.5 ml solution B

15 Cell conveyor:

Run 1: 30 atm Cell 1: comparison A Cell 2: gas 1 (air)

Cell 3: Gas 2 (N2) 20 Cell 4: Gas 3 (Xe: 1 atm / N2: 29 atm)

Archives: Tyrosinase:

Y30G1 ... 4.SP

4 ARCHIVES:

25 Spectrum study: 4/26/91 25 ° C

Cuvettes: 10 min: PARAM: Slot 1 speed 1500 save yes 30 results no CPRG: tyrosinase: ~ 305 nm 61 103984 80 dots => 20 min RUN 16s int

Ymin = ^ max "^

5 Blinds: Tyrosinase: 2 mL solution A + 0.5 mL solution B

Sample: Tyrosinase: 2 ml solution C + 0.5 ml solution B

The cell vehicle:

Run 1: 100 atm Cell 1: Comparison A 10 Cell 2: Gas 1 (Air)

Cell 3: Gas 2 (N2)

Cell 4: Gas 3 (Xe: 1 atm / N2: 29 atm)

Archiving: Y1 = G 1 ... 4.SP

15 4 ARCHIVING:

Protocol: Effect of Pressure on Enzymes Enzyme: Tyrosinase (SIGMA No. T-7755) (Monophenol monooxygenase; polyphenol oxidase; catechol oxidase; monophenol, dihydroxyphenylalanine: oxygen oxidoreductase: EC 20 1.14.18.1)

fungi

Definition of Tvrosinase Unit:

One unit results in the addition of 0.001 per minute A2gQ at pH 6.5 at 25 ° C in a 3 mL reaction mixture containing L-tyrosine-25.

, Tyrosinase effect: 3870 U / mg solid * · 7.1 g solid ==> 27440 units

Store in a dry place at temperatures below 0 ° C

Substrate: L-Tyrosine (SIGMA No. T-3754) 30 L-3- [4-Hydroxyphenyl] Alanine

Free Base (pfs) Crystalline «

Anhydrous Molecule. towards. 181.2 103984 62

Store at room temperature (25 ° C)

Enzyme: β-D-glucosidase (SIGMA No. G-4511) (Emulsion; β-D-glucosidase glucohydrolase EC 3.2.1.21)

Almonds 5 Unit Assay: j One unit releases 1.0 µmol glucose from salicin per minute at pH 5.0 at 37 ° C.

Effect: 22 U / mg solids 12 g solids => 264 units

10 Store dried at 0-5 ° C

Substrate: p-nitrophenyl-β-D-glucopyranoside (SIGMA No. N-7006)

Crystalline

Contains 2.4% solvent Anhydrous Molecule. towards. 301.3

15 Store in a dry place at 0-5 ° C

Experiment 1:

Gases: 1. Air 2. N2 3. Ar 20 4. 02

Pressures: 1. 2 atm (30 psi)

Preparation of the solution: 4/29/91 ready: 4/26/91

Solution A: Sodium phosphate buffer pH 6.8 at 25 ° C

, 2 L deionized water 2 x 141.96 x 0.2 x 245 x 1/1000 = 13.91 g Na2HPO4 2 x 119.96 x 0.2 x 255 x 1/1000 = 12.20 g NaH2PO4 pH tested: 30 Solution B: Tyrosinase solution in Na phosphate buffer (228 U / ml)

14.2 mg T-7755 diluted in 240 mL of Na phosphate buffer pH 6.8 at 25 ° C

103984 63

Solution C: 50 pg / ml L-tyrosine solution in sodium phosphate buffer 10 mg T-3754 diluted in 200 ml Na phosphate buffer pH 6.8 at 25 ° C.

Solution E: 100 µg / ml B-D-glucopyranoside solution in sodium phosphate buffer 5 25 mg N-7006 diluted in 250 ml Na phosphate buffer pH 6.8 at 25 ° C.

Solution F: β-D-glucosidase solution in Na phosphate buffer pH 6.8 (25 ° C) (2.18 units / ml)

24 mg G-4511 diluted in 242 mL Na phosphate buffer pH 6.8 at -10 ° C

Method:

The cell conveyors were removed from the spectrophotometers and the solid cells were mounted.

Acrylic cuvettes were first made by installing a blue silicone stopper, needles 15, and then silicone-optimizing the sides and tops of the cuvettes with a general purpose multipurpose silicone. These were allowed to harden for 48 hours before use. As the silicone cured, the cuvettes were placed vertically and then a tape was applied around the stopper.

Sample Preparation: 4/29/91 The cuvettes were filled for 2 atmospheric experiments of both enzymes.

; Each cuvette was gasified 10 x 10 cm with a suitable gas and allowed to refrigerate for 15 minutes before running. Serum vials were filled with 5 cm of the corresponding enzyme and gasified 10 x 10 cm with a suitable gas before running. The spectrophotometers were set so that the continuous current of each gas could be released into each spectrum at all times. A 100 psimax control element was mounted to read the cuvette pressure and 2 start valves were installed so that the reference cell could be pressurized during the second run. The ball valve was mounted between the cylinder / main housing line and the meter so that gas flow could be monitored directly with spectrophotometers. Control valve 30 was placed in each line to ensure that back pressure would not cause contamination of the gas cylinder liner. The cuvettes held two needles throughout the run. One of the needles was connected to the gas line, while the other was used to introduce the enzyme and then closed. Because of the bulky nature of the assembly, it was impossible to use the spectrophotometer-mounted doors and were therefore removed. A four layer thick black field blanket was fabricated to minimize the amount of light entering each system into the system. When the cuvettes were equilibrated at a temperature in the spectrophotometer, both needles were left

'J

cuvette with 10 cm syringes still attached. Prior to the introduction of the enzyme, one of the syringes was removed while the gas line was connected to the needle. The low gas flow through the line while doing this ensured that there was no oxygen contamination. The lines were above the fluid level.

Another syringe was removed to allow a steady flow through the cuvette.

The enzyme was injected through another needle using a 1 cm syringe. The needle was then blocked, the cuvette pressurized, and driving started.

4/29/91 15 2 atm comparison not pressurized

Tyrosinase: 1. A non-pressurized control or sample.

2. repeat 1 (control cell not pressurized) 4 gases 20 Glucosidase: 1. non-pressurized control or sample.

- - 2nd repeat 1 (reference cell not pressurized) * 4 gases

Spectrum Study: Room Temperature 25 Cuvettes: 10 i 1 PARAM: Slit 1 Speed 1500 save yes; no result 30 TDrive: Tyrosinase: new spectrophotometer (B) 1 305 nm

60 points => 15 min RUN

103984 65 16s int Ymin = 0.0 Ymax = 1 * β-glucosidase: old spectrophotometer (A) 5,400 nm 60 dots => 15 min RUN 16s int

Ymin = ° .0 10 Ymax '^

Blinds: Tyrosinase: 2 mL solution A + 0.5 mL solution B

β-glucosidase: 2 ml solution E + 0.5 ml solution A

Sample: Tyrosinase: 2 ml solution C + 0.5 ml solution B

β-glucosidase: 2 ml solution E + 0.5 ml solution F

15 Archives: 4/29/91

tyrosinase:

Air reference (non-pressurized): 429TYTRL.SP 2 atm (non-pressurized comparison): Y4E1P1G1 ... 7.SP 20 Glucosidase:

Air reference (non-pressurized): 429GLUTR.SP 2 atm (non-pressurized reference): Y4E2P1G1 ... 7.SP Note: The temperature inside the solid sample cell did not remain constant, which directly affects the results obtained. We will remove the fixed 25-cell holders in both spectrophotometers and replace them with cell carriers.

Run 2: 4/30/91 Gases: 1. Air 30 2. N2 3. Ar 4. 02 103984 66

Pressure: 1. 2 atm (30 psi) Sample Preparation:

The solutions prepared on 4/29/91 are reused.

Method: 5 Spectrophotometers were assembled for continuous gas flow as described in Experiment 1. Fixed cell holders were replaced by cell conveyors so that a constant temperature could be maintained using Fischer rotators and Digital control devices. We continued to use black field blankets as they appear to provide adequate light-10 protection.

A non-pressurized gas run was performed on all four gases of all four enzymes. Each cuvette was gasified with 10x10 cm3 of the corresponding gas and cooled for 15 minutes in the refrigerator. The two needles used to gasify the cuvettes were removed when the cuvettes were placed in a spectrophotometer. The enzyme was introduced into the cuvettes using

O

1 cm syringe with attached 20G11 '2 needle. The needle was inserted into the cuvette through a silicone stopper but not immersed in the liquid. This was done to ensure that the introduction of the enzyme into the cuvette was the same as in the pressurized gas run.

Serum vials were filled with 5 emulsions of the respective enzyme and gasified with 10 x 10 cm of suitable gas before running.

The cuvettes:

Tyrosinase: Cuvettes equipped with blue silicone, multi-use silicone and tape were used for this enzyme.

Glucosidase: Only cuvettes containing blue silicone were used for this enzyme.

! * - All cuvettes were pressure tested at 32 psi before use.

Spektrotutkimus:

Cuvettes: 26 30 PARAM: Slot 1 speed 1500 r save yes' '' SS *!

M

103984 There are 67 results

TDrive: Tyrosinase: new spectrophotometer (B) at 25 ° C

305 nm

60 points => 15 min RUN

5 16s int

Ymin _ ^

Ymax = 1.6 β-glucosidase: old spectrophotometer (A) at 35 ° C at 400 nm

10 60 points ==> 15 min RUN

16s int Ymin = 0.0 Ymax = 2> °

Blinds: Tyrosinase: 2 mL solution A + 0.5 mL solution B

15 β-glucosidase: 2 ml solution E + 0.5 ml solution A

Sample: Tyrosinase: 2 ml solution C + 0.5 ml solution B

β-glucosidase: 2 ml solution E + 0.5 ml solution F

Archives: 4/30/91 20 Tyrosinase:

Gas Run (non-pressurized): Y6E1RSG1 ... 4.SP 2 atm (non-pressurized reference): Y6E1P1G1 ... 4.SP 2 atm (reference pressurized): Y7E1P1G1 ... 4.SP Glucosidase:

25 Gas Run (Non-pressurized): Y6E2RSG1 ... 4.SP

2 atm (non-pressurized reference): Y6E2P1G1 ... 4.SP 2 atm (reference pressurized): Y7E2P1G1 ... 4.SP Note: We have chosen to use cuvettes with a blue silicone stopper for the next pressure test. The addition of a general purpose multipurpose silicone and tape appears to reduce the uniformity of the gas-tight closure that the blue silicone has with the cuvette.

103984 68

Experiment 3: 5/1/91

Gases: 1. Air 2. N2 3. Ar 5 4. 02

Pressure: 1. 3 atm (45 psi) Sample Preparation:

The solutions prepared on 4/29/91 are reused.

Method: 10 The same method as in Test 2 4/30/91 is used for this test.

Spektrotutkimus:

Cuvettes: 31 PARAM: Slot 1 speed 1500 15 save yes no result

TDrive: Tyrosinase: new spectrophotometer (B) at 25 ° C

305 nm 60 dots => 15 min RUN 20 16s int •. Ymin = ° '°

Ymax-β-glucosidase: old spectrophotometer (A) at 35 ° C at 400 nm

25 60 points ==> 15 min RUN

16s int Ymin = »-O Ymax = 2> °

Blinds: Tyrosinase: 2 mL solution A + 0.5 mL solution B

30 β-glucosidase: 2 ml solution E + 0.5 ml solution A

~ Z Sample: Tyrosinase: 2 mL solution C + 0.5 mL solution B

β-glucosidase: 2 ml solution E + 0.5 ml solution F

103984 69

Archives: 5/1/91

tyrosinase:

Gas throttle (non-pressurized): Y6E1SRG1 ... 4.SP 5 Gas throttle (non-pressurized): Y7E1SRG1 ... 4.SP

3 atm (reference unpressurized): Y6E1P2G1 ... 4.SP 3 atm (reference pressurized): Y7E1P2G 1 ... 4.SP Glucosidase:

Gas run (not pressurized): Y6E2SRG1 ... 4.SP

10 3 atm (non-pressurized comparison): Y (* E2P2G1 ... 4.SP (USED)

NEW BLOCK) 3 atm (reference pressurized): Y7E2P2G1 ... 4.SP Run 4: 5/3/91

Gases: 1- Air 15 2. Ne 3. Kr 4. Xe

Pressure: 1.2 atm (30 psi)

Preparation of the solution: 5/2/91 20 finished: 4/26/91

. Solution A: Sodium phosphate buffer pH 6.8 at 25 ° C

2 L deionized water 2 x 141.96 x 0.2 x 245 x 1/1000 = 13.91 g Na 2 HPO 4 x 119.96 x 0.2 x 255 x 1/1000 = 12.20 g NaH-> P04 25 pH tested:

Solution B: Tyrosinase solution in Na phosphate buffer (228 U / ml)

7.1 mg T-7755 diluted in 120 mL of Na phosphate buffer pH 6.8 at 25 ° C

Solution C: 50 pg / ml L-tyrosine solution in sodium phosphate buffer 30 mg T-3754 diluted in 200 ml Na phosphate buffer pH 6.8 at 25 ° C.

Solution E: 100 µg / ml β-D-glucopyranoside solution in sodium phosphate buffer 103984 70 12.5 mg N-7006 diluted in 125 ml Na phosphate buffer pH 6.8 at 25 ° C.

Solution F: β-D-glucosidase solution in Na phosphate buffer pH 6.8 (25 ° C) (2.18 units / ml) 5 12 mg G-4511 diluted in 121 mL Na phosphate burst pH 6.8 at 25 ° C A: In this experiment, the same procedure as in experiments 2 and 3 was used, with two exceptions. The needles were immersed in the liquid cuvette after the enzyme was injected into the cuvette and a certain amount of general purpose silicon was placed around the needles to prevent gas from escaping from the cuvette. Spektrotutkimus:

Cuvettes: 20 PARAM: Slot 1 15 Speed 1500 save yes no result

TDrive: Tyrosinase: new spectrophotometer (B) at 25 ° C

305 nm

20 60 points => 15 min RUN

aj. . 16s int

Ymin = °> °

Ymax - .beta.-glucosidase: old spectrophotometer (A) at 35 ° C at 25,400 nm

60 points => 15 min RUN

! 16s int

Ymin = ° '°

Ymax "^

Blinds: Tyrosinase: 2 mL solution A + 0.5 mL solution B

β-glucosidase: 2 ml solution E + 0.5 ml solution A

103984 71

Sample: Tyrosinase: 2 ml solution C + 0.5 ml solution B

B-glucosidase: 2 ml solution E + 0.5 ml solution F

Filing: 5/1/91 5 TDrive to check that the recessed needles do not cause absorption problems.

TDrive: 40 pts, 16 s int, 305 nm: YRONEE1.SP TDrive: 40 pts, 16 s int, 400 nm: GLUCNEE1.SP Tyrosinase:

10 Gas run (non-pressurized): Y6E1SRF5 ... 8.SP

2 atm (comparison unpressurized): Y6E1P1G5 ... 8.SP Glucosidase:

Gas Run (Unpressurized): Y6E2SRG5 ... 8.SP 2 atm (Reference Unpressurized): Y6E2P1G5 ... 8.SP 15 Note: If the needles were in the path of light, they were removed above the liquid level. This was done by turning off the room lights while holding the field cover in place in the spectrophotometer.

Results 20 The results are given as the percent inhibition of gases relative to air inhibition after pressurization to one atmosphere.

For tyrosinase: 2 atm 3 atm 30.6 atm 100 atm (24h) (1h) (-24h) (1h) 25 Air -7.0 & -15.4 -11.6 -37.0 - 4.9 -65.8 -11.1

Xe -76.0 / -86.2 -87.0 -85.7

Kr -84.5 / -93.1

Ar -76.9 / -90.4 -55.8 / -72.1

No -69.0 / -84.5 30 N2 -71.2 / -88.5 -72.1 / -82.6 -78.8 -15.4 -84.7 -20.0 72 103984

The change in relative impact is very large. In the case of xenon, for example, it is 75%. This clearly shows that pressure alone significantly inhibits tyrosinase.

5 For betaglucosidase:

At three atmospheric pressures, the degree of improvement observed was changed as follows: 10 Air 0 (improvement)> -12.5 (inhibition)

Xe +3.1> -15.7

Kr +2.0> -6.2

Ar +1.0> -13.2

Not +1.0> -12.5 15 N2 0> -5.0

The foregoing clearly demonstrates that the reported state of the art inhibitions of hydrolases and other enzymes by noble gases depend on hydrostatic effects.

20

Oxygenation test:

Addition of oxygen to tyrosinase completely reduced the effect of nitrogen. The addition of oxygen lessened the effect of the noble gases, clearly due to the effect of the noble gases on the enzyme through the above molecular properties and simply by the competitive replacement of oxygen from the solution. This evidence distinguishes the effects of noble gases from the effects of nitrogen gas. Increased low oxygen levels also slightly improved tyrosinase activation (as can be expected for an oxygen demanding reaction). But if oxygen was still added, no greater effect was obtained. Thus, reports showing a direct linear relationship between oxygen tension and enzyme effects are inaccurate because they can only describe conditions with limited oxygen.

103984 73

Addition of oxygen to the air caused an increase of up to 6% in the effect of tyrosinase.

Addition of oxygen to the saturated amount in the xenon-saturated solution changed the effect of 5 xenon from 85% inhibition to 50% inhibition.

Addition of oxygen to the argon saturated solution as above changed the effect of argon from 82% inhibition to 12%.

Addition of oxygen as above to the nitrogen-saturated solution completely eliminated the effect of nitrogen from 84% inhibition to zero inhibition.

No effect was observed when oxygen was added to the glucosidase reactions, as might be expected for an oxygen-independent reaction. The addition of oxygen had no effect on the effect of any noble gas on the enzyme, again indicating that the effect of noble gases on the enzyme depends on their molecular properties.

Gas Mixture Test 20 In addition to mixtures of noble gases and nitrogen and oxygen, and Kr and Xe, 1, several mixtures of Ar were tested with either Xe or Kr.

(

Tyrosinase results expressed as inhibition relative to air control: 25 Mixture =% Xe in AR% inhibition =% Kr Arissa% inhibition 0 -75 0 -75 0.1 -72 1.0 -73 5.0 -83 30 10.0 -81 10.0 - 80 50.0 -78 50.0 -78 e 100.0 -86 100.0 -77 103984 74

Similar results were obtained with betaglucosidase, where improvements were observed in all mixtures and were of the same order of magnitude as those obtained with Xe, Kr or Ar, depending on the proximity of the mixture to the pure gases.

5 Protocol: Gas mixtures

Theory: The enzyme effect varies with the gas mixture Gas mixtures: 1. Ar / Xe 2. Ar / Kr 10 1. 0.1% 2. 1.0% 3. 5.0% 4. 10.0% 5. 50.0% 15

Enzyme: Tyrosinase (SIGMA No. T-7755)

(Monophenol monooxygenase; Polyphenol oxidase; Catechol oxidase; Mono phenol, Dihydrophenylalanine: Oxidoreductase: EC

1.14.18.1) 20 Of mushrooms

Determination of Tvrosinase Unit:

One unit causes an increase of 0-001 per minute of A2gQ at pH 6.5 at 25 ° C in a 3 mL reaction mixture containing L-tyrosine.

Effect of tyrosinase: 3870 U / mg solid 25 7.1 mg solid => 27440 units

, Store dried below 0 ° C

·

Lot # 80H9615

Substrate: L-Tyrosine (SIGMA No. T-3754) L-3- [4-Hydroxyphenyl] Alanine Free Base (pfs) Crystalline

Anhydrous Molecule. towards. 181.2 Store at room temperature (25 ° C) 75 103984

Lot # 59F-0478

Enzyme: β-D-glucosidase (SIGMA No. G-4511) (Emulsion: β-D-glucosidase glucohydrolase EC 3.2.1.21)

Almonds 5 Unit Definition:

One unit releases 1.0 μιηοοίΐα glucose from salicin per minute at pH 5.0 at 37 ° C Effect: 22 U / g solids 12 mg solids => 264 units 10 Stored at 0-5 ° C

Lot # 49F-4021

Substrate: p-nitrophenyl-B-D-glucopyranoside (SIGMA No. N-7006)

Crystalline

Contains 2.4% solvent 15 Anhydrous Molecule. towards. 301.3

Store in a dry place at temperatures below 0 ° C

Lot # 129F-5057

Preparation of solution: 20 prepared: 4/15/91

. Solution A: Sodium phosphate buffer pH 6.6 at 25 ° C

1 L Deion Called Water 141.96 x 0.2 x 187.5 x 1/1000 = 5.3 g Na2HPO4 119.96 x 0.2 x 312.5 x 1/1000 = 7.5 g NaH2PO4 25 Fin: 4/17/ 91

Solution B: Tyrosinase solution in Na phosphate buffer (228 U / ml) 14.2 mg T-7755 diluted in 240 mL Na phosphate buffer pH 6.6 at 25 ° C Prepared 4/17/91 30 Solution C: 50 μg / ml L-tyrosine solution sodium phosphate buffer 10 mg T-3754 diluted in 200 ml Na phosphate buffer pH 6.6 at 25 ° C.

76 103984 Fin: 4/10/91

Solution D: Sodium phosphate buffer pH 6.8 at 25 ° C 2 L deionized water 2 x 141.96 x 0.2 x 245 x 1/1000 = 13.91 g of 1 S ^ HPC ^ 5 2 x 119.96 x 0.2 x 255 x 1/1000 = 12.20 g NaH 2 PO 4 Fin: 4/15/91

Solution E: 100 μg / ml β-D-glucopyranoside solution in sodium phosphate buffer 25 mg N-7006 diluted in 250 ml Na phosphate buffer pH 6.8 at 25 ° C.

finished: 4/17/91

Solution F: β-D-glucosidase solution in Na phosphate buffer pH 6.8 (25 ° C) (2.18 units / ml) 24 mg G-4511 diluted in 242 mL of Na phosphate burst pH 6.8 at 15 ° C .

Method: 4/17/91:

Prepare solutions, rinse cuvettes with air after making sure they are gas tight, fill cuvettes and refrigerate, prepare enzyme. . serum vials and finished gas vials.

4/18/91

Gas Blend Finished: 120 cm serum vials were used to mix the injected gases. These vials were rinsed using a vacuum while maintaining one atm of argon in the vial. These vials were cleaned for 20 seconds in this manner. The vials were under mild pressure, 3 which was released through a needle. This is considered to be 120 cm argon. The necessary volumes of Argon and the gas to be mixed (Xe or Kr gases) were then introduced into the vials using a 30 cm syringe and a needle.

30 ”1 103984 77% Argon Xe / Kr 0.1% 120 cm3 0.2 cm3 1.0% 118 cm3 2.4 cm3 5.0% 108 cm3 12 cm3 5 10% 96 cm3 24 cm3 50% 0 cm3 120 cm3

Experiment 1:

We gasified the enzyme using the first vial 10 of the gas mixture using an 8 x 10 cm gas out of the vial. We gasified the cuvette / substrate using a second vial of gas mixture using an 8 x 10 cm gas out of vial. Argon and xenon were removed from the gas cylinders. We filled the enzyme syringe as follows: 15 enzyme gas

With At

Xe Xe 0.1% Ar / Xe Ar 1.0% Ar / Xe Ar 20 5.0% Ar / Xe Ar. Cell conveyor:

Gas Mixture: Ar / Xe, 1 Repetition Run 1: Cell 1: Air

Cell 2: Xe 25 Cell 3: Ar / Xe 0.1%

Cell 4: Ar / Xe 1.0%

Cell 5: Ar / Xe 5.0% Repetition 1: Rl) PARAM: Slot 1 30 speed 1500 save yes no i 78 103984

CPRG: Tyrosinase: 25 ° C

305 nm

'80 points => 20 min RUN

16s int 5 Ymin = °> °

Ymax = 2.0 B-glucosidase: 35 ° C

400 nm 60 dots => 15 min RUN 10 16s int

Ymin =

Ymax "^

Blinds: Tyrosinase: 2 mL solution A + 0.5 mL solution B

β-glucosidase: 2 ml solution E + 0.5 ml solution D

Sample: Tyrosinase: 2 ml solution C + 0.5 ml solution B

β-glucosidase: 2 ml solution E + 0.5 ml solution F

filings:

Tyrosinase: X9R1G1M1 ... 5.SP 20 Glucosidase:

X0R1G1M1 ... 5.SP

Test 1 results:

We found that there was air contamination in the tyrosinase pathway and assumed that this would also be the case in glucosidase pathway. We decided that we would not be able to remove 7 x 8 cm gas mixture from the first vial. Therefore, we decided to make two vials of gas mixture per cuvette for gasification and then two vials of gas mixture per enzyme, which would allow 7 to make 10 x 10 cm per cuvette per vial.

30

Li 103984 79

Experiment 2:

We gasified the second run using 2 vials of gas mixture per cuvette and 2 vials of gas mixture per enzyme. We then proceed by making the last 2 gas mixtures (10% and 50%).

5

Gas mixture: Ar / Xe, 1 repeat

Cell conveyor:

Run 2: Kenno 1: Ar 10 Kenno 2: Xe

Cell 3: Ar / Xe 10.0%

Cell 4: Ar / Xe 50.0%

Cell 5: Air (Repetition 1: R1) 15 PARAM: Slot 1

speed 1500 Asave Y Aprint N

20

CPRG: Tyrosinase: 25 ° C

305 nm 80 dots => 20 min RUN 16s int 25 Ymin = 0.0

Ymax = 1> 6 β-glucosidase: 35 ° C

400 nm 60 dots => 15 min RUN 30 16 s int

Ymin ~ 0.0 Ymax = 1'5 103984 80

Blinds: Tyrosinase: 2 mL solution A + 0.5 mL solution B

β-glucosidase: 2 ml solution E + 0.5 ml solution D

Sample: Tyrosinase: 2 ml solution C + 0.5 ml solution B

β-glucosidase: 2 ml solution E + 0.5 ml solution F

5 Archives:

tyrosinase:

X9R1G1M6 ... 0.SP

glucosidase:

X0R1G1M6 ... 0.SP

10

Test 2 results:

The difference with tyrosinase is that we assume that our new gasification method is appropriate and will continue to gasify in this manner. Therefore, in the future, we will focus on 15 tyrosinases, especially with 10 and 50% gas mixtures.

Run 3: Kenno 1: Ar

Kenno 2: Xe

Cell 3: Ar / Xe 10.0% 20 Cell 4: Ar / Xe 50.0%

Cell 5: Air (Repeat 2: R2)

filings:

tyrosinase:

X9R2G1M6 ... 0.SP

25

We prepared Ar / Kr blends as described above and performed 2 repetitions of 10% and 50% blends.

Driving 4/5: Kenno 1: Ar 30 Kenno 2: Kr

Cell 3: Ar / Xe 10.0%

Kenno 4: Ar / Xe 50.0% 103984 81

Cell 5: Air (repetition 1, 2: R1, R2)

filings:

tyrosinase:

5 X9R1G2M6 ... 0.SP

X9R2G2M6 ... 0.SP

Score:

The results obtained with Ar / Xe look much better than those obtained with Ar / Xe. We continue repeating both mixtures using 10% and 50%. If time permits, we also repeat with 0.1%, 1% and 5% mixtures of both gases or with Tyrosinase only.

15 19.4.91:

Repeats of tyrosinase 2 Ar / Xe on one spectrophotometer and Ar / Kr on another spectrum.

New Spectrum: 20 Run 1: Cell 1: Ar

Kenno 2: Xe

Cell 3: Ar / Xe 10.0%

Cell 4: Ar / Xe 50.0%

Cell 5: Air 25 (Play 3: R3)

Run 2: Cell 1: Ar

Kenno 2: Xe

Cell 3: Ar / Xe 10.0% 30 Cell 4: Ar / Xe 50.0%

Cell 5: Air (Repeat 4: R4) 82 103984

Old spectrum:

Run 1: Cell 1: Ar

Kenno 2: Cr

Cell 3: Ar / Kr 10.0% 5 Cell 4: Ar / Kr 50.0%

Cell 5: Air (Play 3: R3)

Driving 2: Kenno 1: Ar 10 Kenno 2: Cr

Cell 3: Ar / kr 10.0%

Cell 4: Ar / Kr 50.0%

Cell 5: Air (Repeat 4: R4) 15 PARAM: Slot 1

speed 1500 Asave Y Aprint N

20

CPRG: Tyrosinase: 25 ° C

* 305 nm 80 dots => 20 min RUN 16s int 25 Ymin = °> °

Ymax = 2'0

Blinds: Tyrosinase: 2 mL solution A + 0.5 mL solution B

Sample: Tyrosinase: 2 ml solution C + 0.5 ml solution B

30 Archives: Ar / Xe:

X9R3G1M6 ... 0.SP

X9R4G1M6 ... 0.SP

g 83 103984 10 ARCHIVING:

Ar / Kr:

X9R3G2M6 ... 0.SP

X9R4G2M6 ... 0.SP

5 10 ARCHIVES:

Due to lack of time, we were only able to drive:

New spectrum: 10

Run 3: Kenno 1: Ar

Kenno 2: Xe

Cell 3: Ar / Xe 0.1%

Cell 4: Ar / Xe 1.0% 15 Cell 5: Ar / Xe 5.0% (Repeat 2: R2)

Run 4: Kenno 1: Ar

Cell 2: Xe 20 Cell 3: Ar / Xe 0.1%. Cell 4: Ar / Xe 1.0%

Cell 5: Ar / Xe 5.0% (Repeat 3: R3)

Filing: Ar / Xe:

25 X9R2G1M1 ... 0.SP

X9R3G1M1 ... 0.SP

<

: 10 ARCHIVES

Thus, the present invention generally provides a method for controlling enzyme activity by contacting one or more enzymes with a gas containing one or more noble gases or mixtures thereof.

* 103984 84

According to the present invention, the regulated enzyme may be a single, isolated enzyme or one or more enzymes which form a mixture. For example, according to the present invention, a mixture of enzymes can be selectively controlled by inhibiting the effects of one or more enzymes and selectively improving the effects of one or more enzymes.

In addition, the enzyme activity can be adjusted according to the present invention over a temperature range extending from the boiling point of liquid nitrogen (about -200 ° C) to about 120 ° C.

10

According to the present invention, any enzyme can be adjusted using the noble gases present. However, by selecting appropriate conditions for factors such as pH, temperature, pressure, [E] and [S], all enzymes can be specifically inhibited by all noble gases according to the present invention. Further, it will be within the skill of those skilled in the art to use the present disclosure and the above instructions to determine optimum values for pH, temperature, pressure, [E] and [S], a particular enzyme array, or a mixed enzyme array of interest.

The figures of the present specification will now be presented in more detail.

20

Figure 1 depicts the absorption spectra of 2.5 ml tyrosinase (100 μ§ / πιΙ) against 2.5 ml sodium phosphate buffer at pH 6.85.

Figure 2 depicts the absorption spectrum of 2.6 ml of L-tyrosine (100 µg / ml) against 2.5 ml of 25% sodium phosphate buffer at pH 6.85.

i

Figure 3 depicts the absorption spectrum of tyrosinase and L-tyrosine deprived of L-tyrosine at labeled concentrations and labeled pH.

Figure 4 depicts an overview of reaction results run at various concentrations of tyrosinase (40, 60, 80 100 Mg / ml) showing a direct first-order linear (tyrosinase concentration dependence).

103984 85

Figure 5 depicts the inhibition of tyrosinase by xenon at equilibrium ratio (w / w) using labeled concentrations and pH.

Figure 6 illustrates very high inhibition of tyrosinase with xenon at 26 ° C and 5 is estimated to be approximately (area air curve = 11217 μ, xenon = 3037 μ, total = 14254 μ,% total area of air = 78.69%, xenon = 21.31, xenon) total area = 27.08% of air area, air velocity inhibition with xenon = 72.92% This rate of inhibition is very high relative to other conventional enzyme inhibitors.

10

Figure 7 illustrates that at 15 ° C, argon weakly inhibits the equilibrium of the tyrosinase L-tyrosine reaction, xenon has a small but significant inhibitory effect, and neon and krypton have strong effects. Oxygen and air have approximately the same power.

15

Figure 8 illustrates that at 20 ° C, xenon inhibits the tyrosinase-L-tyrosine reaction relatively strongly, while other gases neon, krypton and argon are all potent inhibitors.

Figure 9 illustrates that oxygen has only a moderate inhibitory effect on the tyrosine L-tyrosine reaction.

Figure 10 illustrates a very strong inhibition of the argon tyrosinase L-tyrosine reaction at 20 ° C with almost the same potency of krypton. However, other relevant gases are also potent inhibitors.

Figure 11 illustrates that oxygen has only a weak inhibitory effect on the tyrosinase L-tyrosine reaction at 25 ° C.

Figure 12 illustrates the inhibitory effect of neon, argon, krypton and xenon on the action of tyrosinase at 30 ° C.

103984 86

Figure 13 depicts the inhibitory effect of oxygen on the action of tyrosinase L-tyrosine at 30 ° C.

Figure 14 depicts a standard tyrosinase L-tyrosine reaction run in air showing changes in velocity 5 that are directly comparable to differences in oxygen solubility. The curves obtained with oxygen instead of air are identical.

Figure 15 depicts a temperature-dependent inhibitory effect of neon.

Figure 16 illustrates a direct inverse (negative) relationship between the inhibition capacity and the temperature of krypton tyrosinase.

Figure 17 illustrates that xenon better inhibits the equilibrium of the tyrosinase L-tyrosine reaction than other gases, but reduces the rate of this reaction only as well as krypton and argon. In addition, the shape of the curve varies widely, which is a strong indication of a direct interaction between active site and tyrosinate. Xenon in turn reverses with temperature and has a dynamic effect change in the temperature range 20 ° C to 25 ° C.

Figure 18 illustrates that at 20 ° C, argon, xenon and neon all strongly inhibit the action of tyrosinase, while krypton has a lesser effect. Nitrogen inhibits the reaction quite well, but oxygen has a small effect.

Figure 19 illustrates that at 25 ° C, argon and nitrogen inhibit less than other noble gases, while oxygen improves the reaction. The small difference in the curve between the krypton-xenon and the neon-argon curve pairs is very significant due to the probable relative effects of Van de Waals and the dissolution of oxygen.

Figure 20 illustrates that at 30, oxygen no longer enhances tyrosinase; effect due to reduced solubility. Xenon inhibits the reaction much better than other gases, although all are effective.

103984 87

Figure 21 illustrates the inhibition of tyrosinase activity by neon in which the effect changes between 20 ° C and 25 ° C.

Figure 22 illustrates inhibition of tyrosinase activity by argon, whereby inhibition of argon 5 is better at 20 ° C than neon but less inhibition at 25 ° C than neon. These differences can be considered directly related to solubility / oxygen diffusibility.

Figure 23 illustrates the inhibitory effect of tyrosinase on krypton, where the effect is altered over a temperature range of 20 ° C to 25 ° C. This strongly indicates a direct interaction between the gas and the active site of the enzyme.

Figure 24 illustrates the existence of at least two alterations in the activity of xenons in inhibiting the action of tyrosinase. The first change in action is in the temperature range 15 25 ° C to 30 ° C and the second in the range 30 ° C to 35 ° C. The third change in effect may occur in the range of 20 ° C to 25 ° C. These changes provide strong evidence that there is a direct interaction between the gas and the active site of the enzyme.

Figure 25 illustrates poor inhibition of tyrosine by a solubility mechanism involving removal of oxygen from solution. Air shows the expected dissolution rate pattern as oxygen saturates at 30 ° C.

Figure 26 illustrates the inhibition of tyrosinase activity by nitrogen. Nitrogen is only able to inhibit enzymes by a solubility mechanism involving the removal of oxygen from the solution, but is effective in inhibiting the action of tyrosinase. Change maxima are observed after 20 ° C. The difference between the nitrogen curves and the propellant gas curves represents a possible inhibition of the active site. In most cases, these differences are significant for each gas at least at one temperature.

Figure 27 illustrates the large difference in the effect of xenon in the range 20 ° C to 25 ° C. This corresponds to the optical temperature of the active site of the enzyme.

88 103984

Figure 28 depicts glucose oxidase inhibition by krypton, xenon, argon, nitrogen and neon.

Figure 29 illustrates inhibition of α-glutamyltranspeptidase by krypton, xenon, argon, and a mixture of krypton and xenon.

Figure 30 illustrates the inhibition of aspartate aminotransferase by krypton, xenon, argon and a mixture of krypton and xenon.

Figure 31 illustrates the enhancement of α-D glucosidates by SF SF, argon, nitrogen, neon and oxygen.

Figure 32 illustrates the improvement of ammonia lysate of phenylalanine with neon, oxygen and nitrogen.

15

Figure 33 illustrates the improvement of citrate synthase with xenon and krypton and its inhibition with argon.

Figure 34 illustrates an improvement in phosphoglucose isomerase with xenon, krypton, argon and a mixture of krypton and xenon at 10 ° C.

Figure 35 illustrates the improvement of phosphoglucose isomerase with neon and nitrogen and its inhibition with oxygen at 25 ° C.

Figure 36 illustrates the improvement of S-acetyl Co A synthetase with krypton and a mixture of krypton and xenon at 25 ° C.

Figure 37 illustrates the inhibitory effect of air and nitrogen on B-glucosidase at 100 atmospheric pressure. This effect is simply due to the loss of enzyme damage due to high pressure.

i 103984 89

Figure 38 depicts the inhibitory effect of air and nitrogen on β-glucosidase at 30 atmospheric pressure. This effect is simply due to damage to the enzyme at high pressure.

Figure 39 depicts the inhibitory effect of air, xenon and nitrogen on the action of tyrosinase at 30 atm. pressure. This effect is due to damage to the enzyme at high pressure.

Figure 40 depicts the inhibitory effect of air, xenon and nitrogen on tyrosinase at 100 atm. 10 under pressure. This effect is due to damage to the enzyme at high pressure.

Figure 41 illustrates the effect of enzyme substrate concentrations on the healing and inhibitory effect of noble gases, exemplified by the action of β-D-glucosidase. S1, S2 and S3 represent three different and increasing substrate concentrations. This result is advantageous because it implies that existing biotechnological processes can be modified to reduce the cost of the enzyme and / or substrate or even to facilitate reactions which are currently not possible.

Figure 42 illustrates the different effects of xenon and neon, i.e., they improve. inhibit lactate dehydrogenase at 10 ° C, for example. This is advantageous because it means that a particular enzyme can either be enhanced or inhibited depending on the gas selected.

Figure 43 illustrates that the effect of the present invention is detected even at high temperatures. In particular, at 60 ° C, the action of α-glucosidase is enhanced and inhibited by argon, nitrogen and xenon. Clearly, for this enzyme, argon is a potential enhancer at 60 ° C.

Figure 44 illustrates the effect of xenon at 25 ° C in improving the enzymatic activity of β-glucosidase in immobilized form.

103984 90

As noted above, the enzymatic effects can be adjusted according to the present invention over a wide temperature range from as low as about 120 ° C for liquid nitrogen (about -200 ° C). In addition, the pressure of the gases or gas mixtures used may be as low as the ultra-high vacuum which

O

5 is about 10 atmospheres up to about 100 atmospheres. However, it may be desirable to use even lower or even higher pressures. However, gas pressures of about 10 atmospheres up to about 3 atmospheres are commonly used and 2 gas pressures of about 10 to about 2 atmospheres are most commonly used.

As stated above, the present invention is effective in controlling the enzymatic effect, regardless of the enzyme chosen and the form of the enzyme. The following example is provided to illustrate the effectiveness of the present invention in controlling the enzymatic effects of immobilized enzymes.

15 Protocol: Immobilized enzymes

Theory: The same gases affect either free or immobilized enzymes.

Gases: 1. Air 2. Xenon 20 Enzyme:. β-D-glucosidase (SIGMA) No. G-0898) β-D-glucohydrolase EC 3.2.1.21)

Caldocellum from Saccharolyticum: Recombinant (pfs)

Expressed in E. coli 25 Lyophilized powder

Thermostatic enzyme with a half-life of 38 hours at 70 ° C.

Unit Definition:

One unit liberates 1.0 μηιοο of glucose from saline solution over 30 minutes at pH 5.0 and 37 ° C.

Effect: 46 U / g solids 10.87 g solids => 500 units 103984 91

Store dried at 0.5 ° C Lot # 50H0251

Substrate: p-Nitrophenyl-b-D-glucopyranoside (SIGMA No. N-7006)

Contains 2.4% solvent Anhydrous molecular weight 301.3 Stored at below 0 ° C after storage Lot # 129F-5057 10

Preparation of the solution: ready: 5/21/91

Solution A: Sodium phosphate buffer pH 6.6 at 25 ° C for 1 T. Deionized water 151.96 x 0.2 x 187.5 x 1/1000 = 5.3 g Na 2 HPO 4 119.96 x 0.2 x 312.5 x 1/1000 = 7.5 g NaH 2 PO 4 Fin: 5/21/91

Solution B. 100 µg / mL β-D-glucopyranoside solution in sodium phosphate buffer

25 mg N-7006 diluted in 250 ml sodium phosphate spray pH

6.6 at 25 ° C

Ready: Solution Cl: β-D-glucosidase solution in sodium phosphate buffer pH 6.6 (25 ° C) (2.18 units / ml)

Ig G-0898 solubilized in 21 mL of sodium phosphate buffer pH 6.6 at 25 ° C

(In the case of lyophilized powders for insoluble enzymes, SIGMA recommends suspending the required amount of 5 to 10 mg solids / ml H 2 O and allowing brief hydration). We allowed brief 30 hydration to occur (until dissolution occurs) 400 nm 90 dots => 103984 92 16 s int

Ymin ~

Ymax ~ 5 ^ Blind: 2 ml solution A + 0.5 ml solution Cl

Sample: 2 ml solution B + 0.5 ml solution Cl Filing: G0898G1 ... 4.SP

* TRIAL 2: 5/22/91 Cell conveyor: 10 2 copies

Directions: Cell 1: Aircraft

Cell 2: Air (copy)

Cell 3: Xe 1 Cell 4: Xe (copy) 15 PARAM: Slot 1

speed 1500 Asave Y Aprint N

CPRG: 6-glucosidate: 25 ° C

20,400 nm, 120 dots => 16 s int

Ymin = °> °

Ymax = I · 0 25 Blind: 2 ml solution A + 0.5 ml solution C2 2 ml solution B + 0.5 ml solution C2

Archiving: 2G0898G1 ... 4.SP

To further illustrate the present invention and its effects, kinetic analyzes were performed.

103984 93

Kinetic analyzes

Absorbance curve data, such as those exemplified above, give differences in enzymatic activity across different gas atmospheres, both in terms of yield and rate. Yield can be calculated when equilibrium has been achieved by comparing curve differences and by formal linearization and calculation - which calculations are also necessary to calculate velocity. Velocity differences are calculated using commercially available computer programs (ENZFITTER, GRAFIT, ENZPACK, PEAKFIT) which convert the original uv / vis absorbance data curve (power curve in these examples) into straight lines (linear curve of power curve). These lines can be expressed by a mathematical formula where the slope is approximately velocity and the boundary Y intersept is approximately intake. In addition, this information may be processed to derive Michaelis-Mentenm or other standard biochemical relationships (expressed graphically or mathematically) from which speed and yield can be accurately derived. The examples and figures described in the following 15 exemplify these steps.

Figure 45 depicts uv / vis absorbance curves of β-glucosidase in air at five different substrate concentrations.

Table 1 illustrates the calculations of the Michaelis-Mente enzyme kinetics Vmax and Km and the rates of each reaction. Vmax is the limit speed, Km is the Michael-Mente constant.

»

Figure 46 and Table 2 depict the same as Figure 45, Table 1, however, xenon reactions. It is readily seen that Vmax and Km are higher in the case of xenon.

25th Figure 47, Table 3, Figure 48 and Table 4 illustrate linear transformations of the first order power curve regression rate for the above air and xenon gasified reactions, showing approximation for Michaelis-Mente theories where the Xe / air velocity ratio is the same as above.

30

Figure 49, Figure 50, and Table 5 depict the same first-order velocity approximation regression linearizations for all gases studied in this run. In this 103984 case 94, only xenon had a significant improvement in the power curve analysis, but linearization clearly indicates that other gases improved the reaction.

Figure 51, Table 6, Figure 52, and Table 7 depict data for the first 160 5 seconds of a single tyrosinase assay expressed as a power curve for xenon gasification. Linearised velocity regressions show that Michaelis-Menten declines confirm that xenon strongly inhibits tyrosinase activity.

Table 1 10 _

Gas 1: Air Enzyme kinetics Simple weighing 15 Reduced Chi squared = 0.0000

Variable Αγνό Standard Error V max 0.0028 0.0001 20 Km -2.4539 1.3570 * 'I · n. | I .1. I »II · ........

X Y

[S] Speeds G5 Calculated 25 1 20.0000 0.0032 0.0032 2 40.0000 0.0029 0.0029 3 60.0000 0.0027 0.0029 4 80.0000 0.0030 0.0029 5 100, 0000 0.0029 0.0028 30 The remaining information is graphically represented in the figures as indicated above.

Table 2 95 103984

Gas 5: Xenon Enzyme Kinetics 5 Simple weighing

Reduced Chi squared = 0.0000

Variable Value Standard error 10 _ V max 0.0025 0.0002

Km -3.1783 2.6341

X Y

15 [S] Speeds G1 Calculated 1 20.0000 0.0029 0.0029 2 40.0000 0.0028 0.0027 3 60.0000 0.0025 0.0026 20 4 80.0000 0.0030 0.0026 5 100 , 0000 0.0022 0.0026 25 «* · t

Table 3 96 103984 [S] = 60 micrograms / ml First-order Equation 5 Simple Weighing

Reduced Chi squared = 0.0002

Variable Value Standard Error 10 _

Limit 0.8021 0.0146

Speed constant 0.0032 0.0001

X Y

15 Time Abs Calculated 1 0.0000 0.0471 0.0000 2 15.0000 0.0777 0.0377 3 30.0000 0.1071 0.0737 20 4 45.0000 0.1346 0.1080 5 60.0000 0 , 1626 0.1406 6 75.0000 0.1890 0.1717 7 90.0000 0.2136 0.2014 25 8 105.0000 0.2386 0.22296 9 120.0000 0.2623 0.2566 10 135.0000 0.2849 0.2822 11 150.0000 0.3067 0.3067 12 165.0000 0.33287 0.33300 30 13 180.0000 0.3485 0.3522 14 195.0000 0.3631 0.3734: 15 210 , 0000 0.3881 0.3935 103984 97 16 225.0000 0.4066 0.4128 17 240.0000 0.4244 0.4311 18 255.0000 0.4414 0.4485 19 270.0000 0.4579 0.4652 5 20 285,0000 0.4741 0.4810 21 300.0000 0.4897 0.4961 22 315.0000 0.5044 0.5105 23 330.0000 0.5190 0.5242 24 345.0000 0.5325 0, 5373 10 25 360,0000 0.5448 0.5498 26 375.0000 0.5569 0.5616 27 390.0000 0.5687 0.5730 15 ««

Table 4 98 103984 [S] = 60 micrograms / ml First-order Equation 5 Simple Weighing

Reduced Chi squared = 0.0002

Variable Value Standard Error 10 _

Limit 0.8382 0.0228

Speed constant 0.0045 0.0003

X Y

15 Time Abs [S3] Calculated 1 0.0000 0.1261 0.0000 2 15.0000 0.1590 0.0544 3 30.0000 0.1928 0.1053 20 4 45.0000 0.2217 0.1529 5 60 , 0000 0.2514 0.1794 6 75.0000 0.2811 0.2390 7 90.0000 0.3085 0.2779 8 105.0000 0.33349 0.3143 25 9 120.0000 0.3608 0.3483 10 135,0000 0.3831 0.3801 11 150.0000 0.4065 0.4099 12 165.0000 0.4300 0.4377 13 180.0000 0.4511 0.4637 30 14 195.0000 0.4719 0.4880 15 210,0000 0.4940 0.5108: - 16 225.0000 0.5112 0.5320 99 103984 17 240.0000 0.5311 0.5519 18 255.0000 0.5503 0.5705 19 270.0000 0, 5675 0.5879 20 285.0000 0.5842 0.6042 5 21 300.0000 0.6011 0.6194 22 315.0000 0.6165 0.6336 23 330.0000 0.6313 0.6469 24 345,0000 0 , 6460 0.6593 25 360.0000 0.6588 0.6709 10 26 375.0000 0.6711 0.6818 27 390.0000 0.6834 0.6919 * 9

Table 5 100 103984

Gas 1: Air First order equation 5 Simple weighing

Reduced Chi squared = 0.0002

Variable Value Standard Error 10 _

Limit 0.8021 0.0146

Speed constant 0.0032 0.0001

X Y

15 Time Abs G1 Calculated 1 0.0000 0.0471 0.0000 2 15.0000 0.0777 0.0377 3 30.0000 0.1071 0.0737 20 4 45.0000 0.1346 0.1080. 5 60,0000 0.1626 0.1406 6 75.0000 0.1890 0.1717 7 90.0000 0.2136 0.2014 8 105.0000 0.2386 0.2296 25 9 120.0000 0.2623 0, 2566 r 10 135,0000 0.2849 0.2822 11 150.0000 0.3067 0.3067 12 165.0000 0.33287 0.300 13 180.0000 0.3485 0.3522 30 14 195.0000 0.3691 0.3734 15 210.0000 0.3881 0.3935 16 225.0000 0.4066 0.4128 103984 101 17 240.0000 0.4244 0.4311 18 255.0000 0.4414 0.4485 19 270.0000 0 , 4579 0.4652 20.285.0000 0.4741 0.4810 5 21 300.0000 0.4897 0.4961 22 315.0000 0.5044 0.5105 23 330.0000 0.5190 0.5242 24 345.0000 0.5325 0.5373 25 360.0000 0.5448 0.5498 10 26 375.0000 0.5569 0.5616 27 390.0000 0.5687 0.5730 «102 103984

Table 6

Gas 1: Air Enzyme kinetics 5 Simple weighing

Reduced Chi squared = 0.0000

Variable Value Standard error i 10 _ V max 0.0003 0.0003

Km -17.0661 3.5059

X Y

15 [S] Gas 1: Air Calculated 1 10,0000 0,0018 0,0004 2 20,0000 0,0019 0,0021 3 30,0000 0,0011 0,0007 20 4 40,0000 0,0018 0,0005 5 50,0000 0.0018 0.0005 «i

Table 7 103 103984

Gas 5: Xenon Enzyme Kinetics 5 Simple weighing

Reduced Chi squared = 0.0000

Variable Ano Standard error 10 __ V max 0.0001 0.0003

Km -27.9623 6.3527

X Y

15 [S] Gas 5: Xenon Calculated 1 10,0000 0,0007 -0,0001 2 20,0000 0,0019 -0,0003 3 30,0000 0,0019 0,0017 20 4 40,0000 0,0019 0 , 0004; . 5 50,0000 0.0004 0.0003

Thus, the present invention provides a method for controlling the enzymatic action of any enzyme and in any form. Further, this adjustment can be made over a wide range of temperatures and weights as indicated.

As previously noted, the regulated enzyme or enzymes of the present invention may be in aqueous or organic solution, in immobilized form, in any type of organic matrix dispersion such as a gel. Such solutions, immobilized forms, dispersions or organic matrices are well known to those skilled in the art.

103984 104

Further, according to the present invention, the mixture of enzymes can be adjusted by inhibiting one or more enzymes therein or by improving one or more enzymes therein. Alternatively, it is possible to effect regulation by inhibiting one or more enzymes in the mixture and improving one or more other enzymes therein.

In addition, one skilled in the art is well aware of how to use the present disclosure and the guidance therein to determine optimal pH values, as well as temperature, pressure, [E] and [S], with any particular interest in the enzyme system 10 or enzyme mixture system.

For example, any particular enzyme system or mixture of enzymes of interest, optical pH conditions, pressure, temperature, [E] and [S] can be assured by purifying the enzymes or enzymes using well known reference manuals. For example, the tables of Sigma Chemical Company from 1990 and 1991, and Dixon and Webb's Enzymes. Third Edition (Wiley, 1979). From this information, one skilled in the art can then determine, using the present disclosure, the optimum gas mixture, temperature and pressure to obtain the desired effects in accordance with the present invention.

20

Once the present invention has been described by one of ordinary skill in the art, it will be apparent that many changes and modifications to the above embodiments can be made without departing from the spirit and scope of the present invention.

25

Claims (19)

A process for enhancing enzyme activity, characterized in that at least one enzyme selected from a group consisting of hydrolase enzymes, lyase enzymes, isomerase enzymes and ligase enzymes is reacted with the substrate for this in selected conditions with respect to at pH, temperature, pressure, enzyme concentration (E) and substrate concentration (S), the enzyme being at least part of the reaction time during contact with one or more noble gases and neon, argon, krypton or xenon being used as noble gas. 10 Process according to claim 1, characterized in that the noble gas can comprise a mixture of noble gases. Process according to Claim 1, characterized in that at least one enzyme is present in solution, in immobilized form, in a dispersion or in an organic; matrix. Method according to claim 1, characterized in that at least one of said enzymes is contacted with said gas at a temperature between the temperature of liquid nitrogen and 120 ° C. Process according to claim 4, characterized in that the temperature is 20 - 60 ° C. 25 6. A process according to claim 1, characterized in that at least one enzyme is contacted with said gas under 10 ° - 200 kPa (10 - 2 atm) pressure. 7. A process according to claim 6, characterized in that at least one enzyme 1 is contacted with said gas under 0.1-300 kPa (10-3 atm) pressure. 30 8. A process according to claim 7, characterized in that at least one enzyme is contacted with said gas under 1 - 200 kPa (10 "- 2 atm) pressure. 9. A method according to claim 1, characterized in that at least one enzyme is contacted with said gas during the entire contact time between the enzyme substrate therefor. 5 10. A process according to claim 1, characterized in that said enzyme is a hydrolase enzyme. Process according to claim 1, characterized in that said enzyme is a lyase enzyme. 10 Process according to claim 1, characterized in that said enzyme is an isomerase enzyme. Process according to claim 1, characterized in that said enzyme 15 is a ligase enzyme. A process for regulating enzyme activity in a reaction between an enzyme and its substrate in a liquid medium, wherein the atmosphere surrounding the liquid medium is converted from an enzyme activity inhibitor to an enzyme activity enhancer or, on the contrary, by a noble gas in by changing the temperature. when air is used as side atmosphere, characterized in that at least one enzyme selected from a group consisting of oxidoreductase enzymes, lyase enzymes, isomerase enzymes and ligase enzymes is reacted in a liquid medium with its substrate, whereby the reaction at least a portion of the atmosphere surrounding the liquid medium is replaced by one or more noble gases, which may be neon, argon, krypton or xenon, at a temperature between the temperature of liquid nitrogen and 120 ° C and under a pressure of 10 2 x 10 ^ Pa. 15. A method according to claim 14, characterized in that the control 30 is carried out at a temperature of 20 - 60 ° C. 103984 16. Process according to claim 14, characterized in that the control is carried out under a gas pressure of 0.1 - 300 kPa. Process according to any of claims 14-16, characterized in that the control is carried out under a gas pressure of 1 - 200 kPa. Method according to any of claims 14-17, characterized in that at least one enzyme is present in solution, in immobilized form, in a dispersion or in an organic matrix. 10 19. A process according to claim 15, characterized in that one or more enzymes are regulated by converting the atmosphere from an enzyme activity inhibitor to an enzyme activity enhancer. 15 «